Processing Cartridge and Method for Detecting a Pathogen in a Sample

ABSTRACT

In one embodiment, a multiplex fluid processing cartridge includes a sample well, a deformable fluid chamber, a mixing well with a mixer disposed therein, a lysis chamber including a lysis mixer, an electrowetting grid for microdroplet manipulation, and electrosensor arrays configured to detect analytes of interest. An instrument for processing the cartridge is configured to receive the cartridge and to selectively apply thermal energy, magnetic force, and electrical connections to one or more discrete locations on the cartridge and is further configured to compress the deformable chamber(s) in a specified sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/541,893, filed Aug. 15, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/298,729, filed Oct. 20, 2016 (now abandoned),which is a continuation of U.S. patent application Ser. No. 14/538,565,filed Nov. 11, 2014 (now U.S. Pat. No. 9,498,778), each of which ishereby incorporated by reference.

FIELD OF THE INVENTION

This subject matter of this disclosure relates to systems and methodsfor providing clinical and molecular diagnostics in an integrated,multiplex device that provides sample-to-answer results. In particular,the disclosure relates to a cartridge, to which a sample may be addedand which contains reagents, buffers, and other process materials forperforming a diagnostic assay or other process on the sample, and aninstrument configured to independently process a plurality of suchcartridges.

BACKGROUND OF INVENTION

One major challenge in the area of clinical and molecular diagnostics isthe ability to have a “sample to answer” system that requires minimalsample handling and preparation and minimal requirements for trainedclinical lab personnel. While many systems have been proposed, to datethere are virtually no such commercial systems that adequately meetthese requirements. Aspects of the present invention provide such anintegrated, multiplex system.

SUMMARY OF THE INVENTION

The present invention provides molecular diagnostic methods andcompositions based on the detection of target analytes, includingnucleic acids. The systems described herein are complete integrated“sample to answer” systems, in contrast with current commercial systemsthat require some off chip handling of the sample, generally includingsample extraction (cell lysis, for example), and sample preparationprior to detection. Thus, in accordance with aspects of the currentsystem, a sample is loaded onto a test platform and the target analytesample is extracted, amplified as necessary (for example, when thetarget analyte is a nucleic acid using polymerase chain reaction (PCR)techniques, although isothermal amplification methods can be utilized aswell), and then detected using electrochemical detection, all on amicrofluidic platform, generally referred to herein as a “multiplexcartridge” or a “fluid sample processing cartridge.”

A particular utility of the present system is the ease and rapidity ofthis integrated system. For example, there are no more than 2 operationsrequired before introduction of the sample to the system, which allowsfor both ease of use and no requirement for highly trained labpersonnel. A significant benefit to the present system is also the speedfrom sample to answer, which, in some embodiments, is generally no morethan about 45-90 minutes from sample introduction to reporting of assayresults, with most results being reported in roughly 60-70 minutes orless. This represents a significant advantage to both labs and doctorsrelying on quick analyses for diagnosis and start of appropriatetreatments. In addition, as outlined below, the ability of running notonly multiple tests which are highly multiplexed on a single cartridgebut the ability to analyze multiple cartridges in a completely randomaccess way is a significant advantage in a clinical lab setting. Afurther advantage of the present system is that it can be used forpoint-of-care (POC) diagnostics.

Accordingly, aspects of the present invention are directed to integratedsystems that allow for the detection of target analytes from samples.

For example, aspects of the invention are embodied in a fluid sampleprocessing cartridge comprising a substrate, a sample well formed in thesubstrate, a closure, a deformable fluid chamber supported on thesubstrate, a mixing well formed in the substrate, and a driven mixingapparatus disposed within the mixing well. The sample well is configuredto receive a volume of fluid sample, and the closure is configured to beselectively placed over the sample well. The deformable fluid chamber isconfigured to hold a fluid therein when in an undeformed state and tocollapse upon application of an external compression force to expel atleast a portion of the fluid from the fluid chamber. The deformablefluid chamber is in fluid communication with the sample well via achannel formed in the substrate. The mixing well is in fluidcommunication with the sample well via a channel formed in the substrateand comprises a first peripheral wall and a first floor defining a welland a fluid inlet snorkel extending up a side of the first peripheralwall extending from the channel communicating the mixing well to thesample well and terminating below a top edge of the first peripheralwall. The driven mixing apparatus is constructed and arranged to mix thecontents of the mixing well.

According to further aspects of the invention, the fluid inlet snorkelextends up an outer surface of the first peripheral wall and terminatesat an opening formed in the first peripheral wall.

According to further aspects of the invention, the sample well comprisesa second peripheral wall and a second floor defining a well and a fluidinlet snorkel extending up a side of the second peripheral wall andterminating below a top edge of the second peripheral wall.

According to further aspects of the invention, the mixing well furthercomprises an exit port comprising one or more openings formed in thefloor of the mixing well, wherein the floor tapers downwardly toward theexit port.

According to further aspects of the invention, the driven mixingapparatus comprises a first impeller rotatably disposed within themixing well and a gear configured to be drivingly engaged by a matinggear of an instrument into which the liquid sample processing cartridgeis inserted and to rotate the first impeller when engaged by the matinggear.

According to further aspects of the invention, the sample processingcartridge further comprises a lysis chamber containing a plurality oflysis beads, the lysis chamber being formed in the substrate anddisposed along the channel connecting the mixing well and the samplewell whereby fluid flowing from the sample well to the mixing well willflow through the lysis chamber, and a bead mixer disposed at leastpartially within the lysis chamber and constructed and arranged toagitate the lysis beads and fluid flowing through the lysis chamber.

According to further aspects of the invention, the sample processingcartridge further comprises a first optical interface comprising anenlarged portion of the channel connecting the lysis chamber to thesample well and a second optical interface comprising an enlargedportion of the channel connecting the lysis chamber to the mixing well.

According to further aspects of the invention, the bead mixer comprisesa motor mounted within the substrate and a second impeller disposedwithin the lysis chamber and mounted on an output shaft of the motor.

According to further aspects of the invention, the lysis chamberincludes a fluid inlet and a fluid outlet and further comprises a meshfilter disposed over each of the fluid inlet and the fluid outlet andconfigured to retain the lysis beads within the lysis chamber.

According to further aspects of the invention, the sample processingcartridge further comprises a pressure port formed in the substrate andconfigured to couple the substrate to an external fluid pressure sourceand a channel formed in the substrate connecting the pressure port tothe sample well.

According to further aspects of the invention, the sample processingcartridge further comprises a waste chamber formed in the substrate, thewaste chamber being in fluid communication with the mixing well via achannel formed in the substrate, a fluid exit port formed in thesubstrate, the fluid exit port being in fluid communication with themixing well via a channel formed in the substrate, a first externallyactuatable control valve disposed within the substrate and constructedand arranged to selectively permit or prevent fluid flow from the mixingwell to the waste chamber and a second externally actuatable controlvalve disposed within the substrate and constructed and arranged toselectively permit or prevent fluid flow from the mixing well to thefluid exit port.

According to further aspects of the invention, the sample processingcartridge further comprises a capture chamber disposed along a channelconnecting the mixing well and the waste chamber

According to further aspects of the invention, the sample processingcartridge further comprises a passive valve assembly disposed within thesubstrate and a pressure port formed in the substrate and in pressurecommunication with the passive valve assembly by a pressure conduitformed in the substrate. The passive valve assembly is constructed andarranged to be closed and prevent fluid flow from the mixing well whenpressure within the mixing well is not higher than a threshold pressureand to open and permit fluid flow from the mixing well when pressurewithin the mixing well rises above the threshold pressure. When thepressure port is closed, pressure within the mixing well is allowed toreach the threshold pressure that will open the passive valve assemblyand permit fluid flow from the mixing well, and when the pressure portis open, pressure within the mixing cannot not reach the thresholdpressure so the passive valve assembly is closed.

According to further aspects of the invention, the sample processingcartridge further comprises a lance blister associated with thedeformable fluid chamber. The lance blister is connected or connectableto the associated deformable fluid chamber and contains a bead retainedwithin the lance blister by a breakable septum. The lance blister isconfigured to collapse upon application of an external compression forceto thereby push the bead through the breakable septum.

According to further aspects of the invention, the sample processingcartridge further comprises an external shroud externally enclosing atleast a portion of the cartridge.

According to further aspects of the invention, the sample processingcartridge further comprises a plurality of deformable fluid chambers,and each of the fluid chambers contains one or more substances selectedfrom the group consisting of a lysis buffer, a wash buffer, an oil, arehydration buffer, target capture beads, and a binding buffer.

According to further aspects of the invention, the sample processingcartridge further comprises a first fluid exit port formed in thesubstrate, the first fluid exit port being in fluid communication withthe mixing well via a channel formed in the substrate, a second fluidexit port formed in the substrate, and at least two deformable fluidchambers. One of the two deformable fluid chambers is in fluidcommunication with the mixing well via a channel formed in thesubstrate, and the other of the two deformable fluid chambers is influid communication with the second fluid exit port via a channel formedin the substrate that is different from the channel communicating thefirst fluid exit port with the mixing well.

According to further aspects of the invention, the deformable fluidchamber in fluid communication with the mixing well contains a lysisbuffer, a wash buffer, target capture beads, or a binding buffer, andthe deformable fluid chamber in fluid communication with the secondfluid exit contains an oil or a rehydration buffer.

Further aspects of the invention are embodied in a fluid sampleprocessing cartridge comprising a sample preparation module comprisingand a reaction module. The sample preparation module comprises asubstrate, a sample well formed in the substrate and configured toreceive a volume of fluid sample, a closure configured to be selectivelyplaced over the sample well, a first deformable fluid chamber supportedon the substrate and configured to hold a fluid therein when in anundeformed state and to collapse upon application of an externalcompression force to expel at least a portion of the fluid from thefirst fluid chamber, the first deformable fluid chamber being in fluidcommunication with the sample well via a channel formed in thesubstrate, a mixing well formed in the substrate, the mixing well beingin fluid communication with the sample well via a channel formed in thesubstrate, a driven mixing apparatus disposed within the mixing well andconstructed and arranged to mix the contents of the mixing well, and afirst fluid exit port formed in the substrate, the first fluid exit portbeing in fluid communication with the mixing well via a channel formedin the substrate. The reaction module is attached to the samplepreparation module and is configured to receive a fluid from the samplepreparation module via the fluid exit port formed in the samplepreparation module. The reaction module comprises a top plate comprisinga top surface, a raised wall at least partially circumscribing the topsurface and in fluid sealing contact with a surface of the samplepreparation module to form an interstitial space between the top surfaceand the surface of the sample preparation module, a sample chamberfluidly coupled to the first fluid exit port of the sample preparationmodule, a reagent chamber, and a detection chamber, and a fluidicprocessing panel coupled to a bottom surface of the top plate anddefining a reaction and processing space between the fluidic processingpanel and the top plate. The reaction and processing space is open oropenable to the sample chamber, the reaction chamber, and the detectionchamber.

According to further aspects of the invention, the reaction moduleincludes an inlet port through which fluid sample enters the samplechamber and including a gap between the first fluid exit port of thesample preparation module and the inlet port of the sample chamber, thegap being open to the interstitial space.

According to further aspects of the invention, the first fluid exit portof the sample preparation module comprises an outlet channel formedthrough a frustoconical nipple.

According to further aspects of the invention, reaction module of thefluid sample processing cartridge further comprising an electrosensorarray disposed in each detection chamber.

According to further aspects of the invention, the top plate of thereaction module further comprises one or more bubble traps, each bubbletrap comprising a bubble capture hood open to the reaction andprocessing space and a vent opening open to the interstitial space.

According to further aspects of the invention, the sample preparationmodule further comprises a second deformable fluid chamber supported onthe substrate and configured to hold a fluid therein when in anundeformed state and to collapse upon application of an externalcompression force to expel at least a portion the fluid from the fluidchamber and a second fluid exit port formed in the substrate. The secondfluid exit port is in fluid communication with the second deformablefluid chamber via a channel formed in the substrate, and the reactionand processing space is fluidly coupled to the second fluid exit port ofthe sample preparation module.

According to further aspects of the invention, the mixing well comprisesa peripheral wall and a floor defining a well and a fluid inlet snorkelextending up a side of the peripheral wall extending from the channelcommunicating the mixing well to the sample well and terminating below atop edge of the peripheral wall.

According to further aspects of the invention, the fluid inlet snorkelextends up an outer surface of the peripheral wall and terminates at anopening formed in the peripheral wall.

According to further aspects of the invention, the mixing well furthercomprises an exit port comprising one or more openings formed in thefloor of the mixing well, and the floor tapers downwardly toward theexit port.

According to further aspects of the invention, the driven mixingapparatus comprises a first impeller rotatably disposed within themixing well and a gear configured to be drivingly engaged by a matinggear of an instrument into which the liquid sample processing cartridgeis inserted and to rotate the first impeller when engaged by the matinggear.

According to further aspects of the invention, the sample preparationmodule further comprises a lysis chamber comprising a plurality of lysisbeads, the lysis chamber being formed in the substrate and disposedalong the channel connecting the mixing well and the sample well wherebyfluid flowing from the sample well to the mixing well will flow throughthe lysis chamber, and a bead mixer disposed at least partially withinthe lysis chamber and constructed and arranged to agitate the lysisbeads and fluid flowing through the lysis chamber.

According to further aspects of the invention, the bead mixer comprisesa motor mounted within the substrate and a second impeller disposedwithin the lysis chamber and mounted on an output shaft of the motor.

According to further aspects of the invention, the fluid sampleprocessing cartridge further comprises a first optical interfacecomprising an enlarged portion of the channel connecting the lysischamber to the sample well and a second optical interface comprising anenlarged portion of the channel connecting the lysis chamber to themixing well.

According to further aspects of the invention, the lysis chamberincludes a fluid inlet and a fluid outlet and further comprises a meshfilter disposed over each of the fluid inlet and the fluid outlet andconfigured to retain the lysis beads within the lysis chamber.

According to further aspects of the invention, the sample preparationmodule further comprises a pressure port formed in the substrate andconfigured to couple the substrate to an external fluid pressure sourceand a channel formed in the substrate connecting the pressure port tothe sample well.

According to further aspects of the invention, the sample preparationmodule further comprises a waste chamber formed in the substrate, thewaste chamber being in fluid communication with the mixing well via achannel formed in the substrate, a first externally actuatable controlvalve disposed within the substrate and constructed and arranged toselectively permit or prevent fluid flow from the mixing well to thewaste chamber, and a second externally actuatable control valve disposedwithin the substrate and constructed and arranged to selectively permitor prevent fluid flow from the mixing well to the exit port.

According to further aspects of the invention, the sample preparationmodule further comprises a capture chamber disposed along a channelconnecting the mixing well and the waste chamber.

According to further aspects of the invention, the sample preparationmodule further comprises a passive valve assembly disposed within thesubstrate and constructed and arranged to be closed and prevent fluidflow from the mixing well when pressure within the mixing well is nothigher than a threshold pressure and to open and permit fluid flow fromthe mixing well when pressure within the mixing well rises above thethreshold pressure and a pressure port formed in the substrate and inpressure communication with the passive valve assembly by a pressureconduit formed in the substrate. When the pressure port is closed,pressure within the mixing well is allowed to reach the thresholdpressure that will open the passive valve assembly and permit fluid flowfrom the mixing well, and when the pressure port is open pressure withinthe mixing well cannot reach the threshold pressure so the passive valveassembly is closed.

According to further aspects of the invention, the sample preparationmodule further comprises a lance blister associated with the deformablefluid chamber. The lance blister is connected or connectable to theassociated deformable fluid chamber and contains a bead retained withinthe lance blister by a breakable septum. The lance blister is configuredto collapse upon application of an external compression force to therebypush the bead through the breakable septum.

According to further aspects of the invention, an external shroudexternally encloses at least a portion of the cartridge.

According to further aspects of the invention, the sample preparationmodule further comprises a plurality of deformable fluid chambers, andeach of the fluid chambers contains a substance selected from the groupconsisting of a lysis buffer, a wash buffer, an oil, a rehydrationbuffer, target capture beads, and a binding buffer.

Additional aspects of the invention are embodied in an instrumentconfigured to process a fluid sample processing cartridge including adeformable fluid chamber supported on a planar substrate and configuredto hold a fluid therein when in an undeformed state and to collapse uponapplication of an external compression force to expel at least a portionof the fluid from the fluid chamber. The instrument comprises acartridge carriage assembly a cartridge carriage assembly configured toreceive and hold a fluid sample processing cartridge inserted into theinstrument. A heating and control assembly is disposed adjacent thecartridge carriage assembly and is configured for movement with respectto the cartridge carriage assembly between a first position not inoperative contact with the cartridge carried within the cartridgecarriage assembly and a second position in operative contact with thecartridge carried within the cartridge carriage assembly. One or moremovable magnet assemblies are each mounted for movement with respect tothe cartridge independently of the heating and control assembly betweena first position applying substantially no magnetic force to thecartridge and a second position applying magnetic force to correspondingdiscrete portions of the cartridge. A cam block assembly is configuredfor powered movement and is operatively coupled to the heating andcontrol assembly for converting powered movement of the cam blockassembly into movement of the heating and control assembly with respectto the cartridge carriage assembly between the first position of theheating and control assembly and the second position of the heating andcontrol assembly. The cam block assembly is operatively coupled to theone or more moveable magnet assemblies for converting powered movementof the cam block assembly into movement of each magnet assembly withrespect to cartridge carriage assembly between the first position of themagnet assembly and the second position of the magnet assembly. Adeformable chamber compression assembly is configured to selectivelyapply an external compression force to the deformable fluid chamber tocollapse the deformable chamber and expel at least a portion of thefluid from the fluid chamber.

According to further aspects of the invention, the heating and controlassembly comprises one or more heater assemblies configured to apply athermal gradient to corresponding discrete portions of the cartridgewhen the heating and control assembly is in the second position and aconnector board including one or more electrical connector elementsconfigured to effect an electrical connection between the instrument andthe cartridge when the heating and control assembly is in the secondposition.

According to further aspects of the invention, the deformable chambercompression assembly comprises a cam follower plate configured forpowered movement in a first direction that is generally parallel to theplane of the substrate and a compression mechanism associated with thedeformable chamber of the cartridge and configured to apply a forcecompressing the chamber against the substrate by movement in a seconddirection having a component that is generally normal to the plane ofthe substrate. The cam follower plate is operatively coupled to thecompression mechanism to convert movement of the cam follower plate inthe first direction into movement of the compression mechanism in thesecond direction to thereby apply an external compression force to thechamber.

According to further aspects of the invention, the instrument furthercomprises a pneumatic pump and a pneumatic port connected to thepneumatic pump, wherein the pneumatic port is configured to couple thepneumatic pump to a pressure port of the fluid sample processingcartridge when the cartridge is inserted into the instrument.

According to further aspects of the invention, the instrument furthercomprises an optical detector configured to detect fluid flow through apart of the fluid sample processing cartridge.

According to further aspects of the invention, the fluid sampleprocessing cartridge includes a driven mixing apparatus including adrive gear, and the instrument further comprises a mixing motor assemblyincluding a powered driving gear. The mixing motor is moveable between afirst position in which the driving gear is not engaged with the drivegear of the driven mixing apparatus and a second position in which thedriving gear is operatively engaged with the drive gear to actuate thedriven mixing apparatus. The cam block assembly is operatively coupledto the mixing motor assembly for converting powered movement of the camblock assembly into movement of the mixing motor assembly between thefirst position of the mixing motor assembly and the second position ofthe mixing motor assembly.

According to further aspects of the invention, the instrument furthercomprises a heater cooling assembly comprising a fan and a cooling ductconfigured to direct air flow from the fan to a portion of one of theheater assemblies.

According to further aspects of the invention, the cartridge carriageassembly comprises a cartridge holder configured to hold a cartridgeinserted therein, a cartridge latch biased into a cartridge-latchingposition and configured to latch onto a cartridge inserted into thecartridge holder to retain the cartridge within the cartridge holder,and a cartridge eject mechanism configured to automatically push acartridge at least partially out of the cartridge holder when thecartridge latch is released from a cartridge-latching position.

According to further aspects of the invention, the heating and controlassembly comprises a support plate on which the one or more heaterassemblies and the connector board are supported. The support plate ismounted in a constrain configuration preventing horizontal movement ofthe support plate but permitting vertical movement of the support plateto enable movement of the heating and control assembly between its firstand second positions.

According to further aspects of the invention, the heater assemblies ofthe heating and control assembly comprises a resistive heating elementattached to the connector board and a heat spreader comprising athermally-conductive material thermally coupled to the resistive heatingelement.

According to further aspects of the invention, one of the heaterassemblies of the heating and control assembly comprises athermoelectric element, a heat spreader comprising athermally-conductive material thermally coupled to the thermoelectricelement, and a heat sink including a panel that is in thermal contactwith the thermoelectric element and a plurality of heat-dissipatingrods.

According to further aspects of the invention, the electrical connectorelements of the connector board of the heating and control assemblycomprise a plurality of connector pin arrays, each connector pin arraycomprising a plurality of pogo pins.

According to further aspects of the invention, one of the movable magnetassemblies comprises a magnet holder mounted on a spindle so as to berotatable about the spindle between the first position and the secondposition of the magnet assembly, a magnet supported on the magnetholder, an actuator bracket extending from the magnet holder, and atorsion spring configured to bias the magnet holder to a rotationalposition corresponding to the first position of the magnet assembly.

According to further aspects of the invention, one of the movable magnetassemblies comprises a magnet holder frame mounted on a spindle so as tobe rotatable about the spindle between the first position and the secondposition of the magnet assembly, a magnet array disposed within themagnet holder frame, a focusing magnet disposed within an opening formedin the magnet holder frame and configured to focus magnetic forces ofthe magnet array, an actuator bracket extending from the magnet holderframe, and a torsion spring configured to bias the magnet holder frameto a rotational position corresponding to the first position of themagnet assembly.

According to further aspects of the invention, the cam block assembly isoperatively coupled to each movable magnet assembly by a magnet actuatorcoupled at one portion thereof to the cam block assembly so as to bemoveable by powered movement of the cam block assembly and including atab configured to be engageable with the actuator bracket of each magnetassembly as the magnet actuator is moved with the cam block assembly tocause corresponding rotation of the magnet assembly from the firstposition to the second position.

According to further aspects of the invention, the cam block assemblycomprises a cam frame, a cam block motor coupled to the cam frame andconfigured to effect powered movement of the cam frame, and first andsecond cam rails attached to the cam frame. Each of the cam rails hastwo cam slots. The cam block assembly is operatively coupled to theheating and control assembly by cam followers extending from the heatingand control assembly into the cam slots such that movement of the camframe and the cam rails with respect to the heating and control assemblycauses corresponding relative movement between the cam followers and thecam slots to move the cam followers between respective first segments ofthe cam slots corresponding to the first position of the heating andcontrol assembly and respective second segments of the cam slotscorresponding to the second position of the heating and controlassembly.

According to further aspects of the invention, the cam frame comprises afirst longitudinal spar extending along one side of the heating andcontrol assembly, a second longitudinal spar extending along an oppositeside of the heating and control assembly, and a cross spar extendingbetween the first and second longitudinal spars. Each cam rail isattached to one of the first and second longitudinal spars.

According to further aspects of the invention, the compression mechanismof the deformable chamber compression assembly comprises a cam armhaving a cam surface and mounted so as to be pivotable about one end ofthe cam arm and a compression pad disposed at an opposite end of the camarm, wherein the cam arm is pivotable between a first position in whichthe compression pad does not contact the associated deformable chamberand a second position in which the compression pad applies a compressiveforce to the associated deformable chamber to at least partiallycollapse the chamber.

According to further aspects of the invention, the deformable chambercompression assembly further comprises a cam arm plate, and the cam armof the compression mechanism is pivotably mounted within a slot formedin the cam arm plate for pivotable movement of the cam arm with respectto the cam arm plate. The cam surface of the cam arm projects out of theslot above a surface of the cam arm plate. The cam follower plate isoperatively coupled to the compression mechanism by a cam followerelement of the cam follower plate that is engaged with the cam surfaceof the compression mechanism during movement of the cam follower platewith respect to the cam arm plate to cause the cam arm to pivot from itsfirst position to its second position.

According to further aspects of the invention, the cam follower platecomprises a cam groove that receives the cam surface of the cam armprojecting above the surface of the cam arm plate, and the cam followerelement comprises a follower ridge disposed within the cam groove thatcontacts the cam surface as the cam follower plate moves with respect tothe cam arm plate to cause the cam arm to pivot from its first positionto its second position.

According to further aspects of the invention, the instrument furthercomprises a plurality of compression mechanisms, each comprising a camarm pivotably mounted within a slot formed in the cam arm plate and acam arm surface, and the cam follower plate comprises a plurality of camgrooves, each cam groove being associated with at least one of thecompression mechanisms and each cam groove including a follower ridgedisposed within the cam groove that contacts the cam surface of theassociated compression mechanism as the cam follower plate moves withrespect to the cam arm plate to cause the cam arm of the associatedcompression mechanism to pivot from its first position to its secondposition.

According to further aspects of the invention, the sample processingcartridge includes a plurality of deformable fluid chambers, and thedeformable chamber compression assembly comprises a plurality ofcompression mechanisms. Each compression mechanism is associated withone of the deformable fluid chambers, and the cam follower plate isoperatively coupled to the compression mechanisms to convert movement ofthe cam follower plate in the first direction into movement of each ofthe compression mechanisms in the second direction to thereby apply anexternal compression force to each of the associated chambers in aspecified sequence

According to further aspects of the invention, the fluid sampleprocessing cartridge includes an externally-actuatable control valveconfigured to selectively control fluid flow by permitting fluid flowthrough the valve when not externally actuated and preventing fluid flowthrough the valve when externally actuated. The instrument furthercomprises a valve actuator compression mechanism associated with theexternally-actuatable control valve of the sample processing cartridgeand configured to actuate the associated externally-actuatable controlvalve by movement in a second direction having a component that isgenerally normal to the plane of the substrate. The cam follower plateis operatively coupled to the valve actuator compression mechanism toconvert movement of the cam follower plate in the first direction intomovement of the valve actuator compression mechanism in the seconddirection to thereby actuate the associated externally-actuatablecontrol valve.

Other features and characteristics of the subject matter of thisdisclosure, as well as the methods of operation, functions of relatedelements of structure and the combination of parts, and economies ofmanufacture, will become more apparent upon consideration of thefollowing description and any appended claims with reference to theaccompanying drawings, all of which form a part of this specification.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the subjectmatter of this disclosure. In the drawings, like reference numbersindicate identical or functionally similar elements.

FIG. 1 is a top perspective view of a multiplex cartridge embodyingaspects of the present invention.

FIG. 2 is a top plan view of the multiplex cartridge.

FIG. 3 is a top plan view of the multiplex cartridge annotated withidentifying labels.

FIG. 4 is an exploded perspective view of the multiplex cartridge.

FIG. 5 is a partial perspective view in cross-section of a deformablefluid compartment (or blister) of the multiplex cartridge.

FIG. 6 is a perspective detail of a sample well and a sample cap of themultiplex cartridge.

FIG. 7 is a perspective, cross sectional view of the sample well alongthe line 7-7 in FIG. 2.

FIG. 8A is a perspective detail of a mixing well and mixer of themultiplex cartridge.

FIG. 8B is a perspective detail of an alternate mixing well of themultiplex cartridge.

FIG. 8C is a top plan view of the mixing well of FIG. 8C.

FIG. 9A is a cross sectional view of the mixing well and mixer along theline 9-9 in FIG. 2.

FIG. 9B is cross sectional view of the alternate mixing well of FIGS. 8Band 8C and an alternate mixer disposed therein.

FIG. 10 is a perspective detail of a passive valve of the multiplexcartridge.

FIG. 11 is a perspective, cross sectional view of the passive valvealong the line 11-11 in FIG. 2.

FIG. 12 is a perspective, cross sectional view of a lysis chamber andbead mixer along the line 12-12 in FIG. 2.

FIG. 13 is a perspective, cross sectional view of an active valveassembly along the line 13-13 in FIG. 2.

FIG. 14 is a perspective, cross sectional view of the active valve,wherein the valve is actuated by an external valve actuator.

FIG. 15 is a top plan view of a sample preparation module of themultiplex cartridge.

FIGS. 16-23 show top plan views of the sample preparation module, eachshowing a different step of a sample preparation process performedwithin the module.

FIG. 24 is a top perspective view of a top plate of a reaction module ofthe multiplex cartridge.

FIG. 25 is a bottom perspective view of the top plate.

FIG. 26 is a top plan view of the top plate.

FIG. 27 is a bottom plan view of the top plate.

FIG. 28 is perspective, cross sectional view of the reaction modulealong the line 28-28 in FIG. 24.

FIG. 29 perspective, cross sectional view of the reaction module alongthe line 29-29 in FIG. 24.

FIG. 30 is a perspective detail of a fluid inlet of the reaction module.

FIG. 31 is a partial cross sectional view along the line 31-31 in FIG.26.

FIG. 32. is a front view of an instrument embodying aspects of theinvention.

FIG. 33 is a front perspective view of a control console of theinstrument.

FIG. 34 is a front perspective view of a processing module of theinstrument.

FIG. 35 is a front perspective view of the processing module with oneside wall of the module removed to show internal components of theprocessing module.

FIG. 36 is a rear perspective view of the processing module with oneside wall and the rear wall of the module removed to show internalcomponents of the processing module.

FIG. 37 is a front perspective view of the processing module with oneside wall and one rear wall of the module removed and with oneprocessing bay of the processing module exploded from the module.

FIG. 38 is a front, right-side perspective view of a processing bayembodying aspects of the present invention.

FIG. 39 is a front, left-side perspective view of the processing bay.

FIG. 40 is a rear, right-side perspective view of the processing bay.

FIG. 41 is a front, right-side, exploded perspective view of theprocessing bay.

FIG. 42 is an exploded perspective view of the cartridge processingassembly of the processing bay.

FIG. 43 is an exploded perspective view of a heating and controlassembly of the cartridge processing assembly.

FIG. 44 is a top plan view of a connector PCB and magnets of the heatingand control assembly of the cartridge processing assembly.

FIG. 45 is an exploded perspective view of a detection Peltier heaterassembly of the heating and control assembly.

FIG. 46 is an exploded perspective view of a cartridge carriage assemblyof the cartridge processing assembly.

FIG. 47 is an exploded perspective view of the cam frame assembly of thecartridge processing assembly.

FIG. 48 is a perspective, cross-sectional view of the cam frame and amagnet actuator of the cartridge processing assembly.

FIG. 49A is a top perspective view of a sample preparation magnetassembly of the cartridge processing assembly.

FIG. 49B is a top perspective view of a cartridge magnet assembly of thecartridge processing assembly.

FIG. 50A is a perspective view of a mixing motor assembly of thecartridge processing assembly.

FIG. 50B is an exploded perspective view of the mixing motor assembly.

FIG. 51 is an exploded prospective view of a blister compressionmechanism assembly of the processing bay.

FIG. 52 is a partial bottom plan view of a cam arm plate showingcompression pads of an array of compression mechanisms.

FIG. 53 is a top perspective view of the compression mechanisms of thearray isolated from the cam arm plate.

FIG. 54 is a bottom perspective view of the compression mechanisms ofthe array isolated from the cam arm plate.

FIG. 55A is an exploded perspective view of a single fluid blistercompression mechanism.

FIG. 55B is an exploded prospective view of a single lance blistercompression mechanism.

FIG. 55C is an exploded perspective view a single valve actuatorcompression mechanism.

FIG. 56 is a bottom plan view of a cam follower plate of the blistercompression mechanism assembly.

FIG. 57 is a bottom perspective view of the cam follower plate.

FIG. 58 is a bottom plan view of a fluidic processing panel of thereaction module.

FIG. 59 is a top plan view of the fluidic processing panel.

FIG. 60 is a flow chart illustrating an exemplary process that can beperformed in the fluidic processing panel.

DETAILED DESCRIPTION OF THE INVENTION

While aspects of the subject matter of the present disclosure may beembodied in a variety of forms, the following description andaccompanying drawings are merely intended to disclose some of theseforms as specific examples of the subject matter. Accordingly, thesubject matter of this disclosure is not intended to be limited to theforms or embodiments so described and illustrated.

Unless defined otherwise, all terms of art, notations and othertechnical terms or terminology used herein have the same meaning as iscommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. All patents, applications, published applicationsand other publications referred to herein are incorporated by referencein their entirety. If a definition set forth in this section is contraryto or otherwise inconsistent with a definition set forth in the patents,applications, published applications, and other publications that areherein incorporated by reference, the definition set forth in thissection prevails over the definition that is incorporated herein byreference.

Unless otherwise indicated or the context suggests otherwise, as usedherein, “a” or “an” means “at least one” or “one or more.”

This description may use relative spatial and/or orientation terms indescribing the position and/or orientation of a component, apparatus,location, feature, or a portion thereof. Unless specifically stated, orotherwise dictated by the context of the description, such terms,including, without limitation, top, bottom, above, below, under, on topof, upper, lower, left of, right of, in front of, behind, next to,adjacent, between, horizontal, vertical, diagonal, longitudinal,transverse, radial, axial, etc., are used for convenience in referringto such component, apparatus, location, feature, or a portion thereof inthe drawings and are not intended to be limiting.

Furthermore, unless otherwise stated, any specific dimensions mentionedin this description are merely representative of an exemplaryimplementation of a device embodying aspects of the invention and arenot intended to be limiting.

RELATED APPLICATIONS

This application is related to U.S. patent application Ser. No.14/062,860 and U.S. patent application Ser. No. 14/062,865 (U.S. PatentApplication Publication No. 2014-0194305), the respective disclosures ofwhich are hereby incorporated by reference.

INTRODUCTION

In general, the system includes two components: the multiplex cartridge,into which the sample is loaded and which contains various reagents,buffers, and other processing materials for performing the desired assayor other procedure, and the processing instrument into which thecartridge is inserted to perform the sample processing and finaldetection of the target analytes.

In various embodiments, the microfluidic platform relies on theformation of microdroplets and the ability to independently transport,merge, mix and/or process the droplets. In various embodiments, suchmicrodroplet operations are performed using electrical control ofsurface tension (i.e., electrowetting). In general, liquid samples arecontained within a microfluidic device, known as a processing module,between two parallel plates. One plate—referred to as the fluidicprocessing panel—contains etched drive electrodes on its surface whilethe other plate contains either etched electrodes or a single,continuous plane electrode that is grounded or set to a referencepotential (“biplanar electrowetting”). Hydrophobic insulation covers theelectrodes and an electric field is generated between electrodes onopposing plates. This electric field creates a surface-tension gradientthat causes a droplet overlapping the energized electrode to movetowards that electrode. In some embodiments, the active electrowettingelectrodes may be adjacent and on the same plane as the neighboringground reference electrode, which is referred to as “coplanarelectrowetting”. Through proper arrangement and control of theelectrodes, a droplet can be transported by successively transferring itbetween adjacent electrodes. The patterned electrodes can be arranged ina two dimensional array so as to allow transport of a droplet to anylocation covered by that array. The space surrounding the droplets maybe filled with a gas such as air or an immiscible fluid such as oil,with immiscible oils being preferred in many embodiments of the presentinvention.

As the droplets containing the target analytes move across the surface,they can pick up reagents and buffers. For example, when dried reagentsare placed on the surface (generally described herein as printed circuitboard, although as will be appreciated by those in the art, additionalsurfaces can be used), a droplet moving through that zone will pick upand dissolve the reagent for use in a biological process, such as PCRamplification. In addition, as more fully described below, addition froma sample preparation module positioned above the substrate, allows forspecific addition of buffers and other reagents such as wash buffers,etc., as well as preparation, e.g., lysis, purification, dissolution,etc., of the sample prior to transferring the sample to the microfluidicplatform.

Aspects of the present invention also involve the use of electrochemicaldetection of analytes of interest. Suitable electrochemical detectionsystems are described in U.S. Pat. Nos. 4,887,455; 5,591,578; 5,705,348;5,770,365; 5,807,701; 5,824,473; 5,882,497; 6,013,170; 6,013,459;6,033,601; 6,063,573; 6,090,933; 6,096,273; 6,180,064; 6,190,858;6,192,351; 6,221,583; 6,232,062; 6,236,951; 6,248,229; 6,264,825;6,265,155; 6,290,839; 6,361,958; 6,376,232; 6,431,016; 6,432,723;6,479,240; 6,495,323; 6,518,024; 6,541,617; 6,596,483; 6,600,026;6,602,400; 6,627,412; 6,642,046; 6,655,010; 6,686,150; 6,740,518;6,753,143; 6,761,816; 6,824,669; 6,833,267; 6,875,619; 6,942,771;6,951,759; 6,960,467; 6,977,151; 7,014,992; 7,018,523; 7,045,285;7,056,669; 7,087,148; 7,090,804; 7,125,668; 7,160,678; 7,172,897;7,267,939; 7,312,087; 7,381,525; 7,381,533; 7,384,749; 7,393,645;7,514,228; 7,534,331; 7,560,237; 7,566,534; 7,579,145; 7,582,419;7,595,153; 7,601,507; 7,655,129; 7,713,711; 7,759,073; 7,820,391;7,863,035; 7,935,481; 8,012,743; 8,114,661 and U.S. Pub. No. 2012/01 81186, the respective disclosures of which are expressly incorporatedherein by reference.

In various embodiments processed target analyte droplets are transportedto a detection zone on the fluidic processing panel, where they arespecifically captured on individual detection electrodes, using systemsdescribed in numerous patents above with specific reference to U.S. Pat.Nos. 7,160,678, 7,393,645, and 7,935,481. This detection system relieson the use of label probes (in the case of nucleic acids) containingelectrochemically active labels, such that the presence of the targetanalyte results in a positive signal, allowing detection of thepathogen, disease state, etc.

Samples

Aspects of the invention provide systems and methods for the detectionof target analytes in samples to diagnose disease or infection bypathogens (e.g. bacteria, virus, fungi, etc.). As will be appreciated bythose in the art, the sample solution may comprise any number of things,including, but not limited to, bodily fluids (including, but not limitedto, blood, urine, serum, plasma, cerebrospinal fluid, lymph, saliva,nasopharyngeal samples, anal and vaginal secretions, feces, tissuesamples including tissues suspected of containing cancerous cells,perspiration and semen of virtually any organism, with mammalian samplesbeing preferred and human samples being particularly preferred);environmental samples (including, but not limited to, air, agricultural,water and soil samples, environmental swabs and other collection kits);biological warfare agent samples; food and beverage samples, researchsamples (i.e., in the case of nucleic acids, the sample may be theproducts of an amplification reaction, including both target and signalamplification as is generally described in WO/1999/037819, thedisclosure of which is hereby incorporated by reference, such as PCRamplification reaction); purified samples, such as purified genomic DNA,RNA, proteins, etc.; raw samples (bacteria, virus, genomic DNA, etc.);as will be appreciated by those in the art, virtually any experimentalmanipulation may have been done on the sample.

The multiplex cartridge may be used to detect target analytes in patientsamples. By “target analyte” or “analyte” or grammatical equivalentsherein is meant any molecule or compound to be detected and that canbind to a binding species, defined below. Suitable analytes include, butare not limited to, small chemical molecules such as environmental orclinical chemical or pollutant or biomolecule, including, but notlimited to, pesticides, insecticides, toxins, therapeutic and abuseddrugs, hormones, antibiotics, antibodies, organic materials, etc.Suitable biomolecules include, but are not limited to, proteins(including enzymes, immunoglobulins and glycoproteins), nucleic acids,lipids, lectins, carbohydrates, hormones, whole cells (includingprokaryotic (such as pathogenic bacteria) and eukaryotic cells,including mammalian tumor cells), viruses, spores, etc.

In one embodiment, the target analyte is a protein (“target protein”).As will be appreciated by those in the art, there are a large number ofpossible proteinaceous target analytes that may be detected using thepresent invention. By “proteins” or grammatical equivalents herein ismeant proteins, oligopeptides and peptides, derivatives and analogs,including proteins containing non-naturally occurring amino acids andamino acid analogs, and peptidomimetic structures. The side chains maybe in either the (R) or the (S) configuration. In a preferredembodiment, the amino acids are in the (S) or L-configuration. Asdiscussed below, when the protein is used as a binding ligand, it may bedesirable to utilize protein analogs to retard degradation by samplecontaminants. Particularly preferred target proteins include enzymes;drugs, cells; antibodies; antigens; cellular membrane antigens andreceptors (neural, hormonal, nutrient, and cell surface receptors) ortheir ligands.

In a preferred embodiment, the target analyte is a nucleic acid (“targetnucleic acid”). The present system finds use in the diagnosis ofspecific pathogens exogenous to a patient such as bacteria and viruses,as well as the diagnosis of genetic disease, such as single nucleotidepolymorphisms (SNPs) that cause disease (e.g. cystic fibrosis) or arepresent in disease (e.g. tumor mutations).

As will be appreciated by those in the art, the present invention relieson both target nucleic acids and other nucleic acid components likecapture probes and label probes used in the detection of the targetnucleic acids. By “nucleic acid” or “oligonucleotide” or grammaticalequivalents herein means at least two nucleotides covalently linkedtogether. A nucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs can be included as primers or probes that may havealternate backbones, comprising, for example, phosphoramide (Beaucage etal., Tetrahedron 49(10).T 925 (1993) and references therein; Letsinger,J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579(1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al,Chem. Left. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 1 10:4470(1988); and Pauwels et al., Chemica Scripta 26: 141 91986)),phosphorothioate (Mag et al., Nucleic Acids Res. 19: 1437 (1991); andU.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al, J. Am. Chem.Soc. 1 1 1:2321 (1989), O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc. 1 14: 1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature380:207 (1996), all of which are incorporated by reference). Otheranalog nucleic acids include those with positive backbones (Denpcy etal., Proc. Natl. Acad. Sci. USA 92:6097 (1995); those with bicyclicstructures including locked nucleic acids, Koshkin et al., J. Am. Chem.Soc. 120: 13252-3 (1998); non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216, 141 and 4,469,863; Kiedrowshi et al.,Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am.Chem. Soc. 1 10:4470 (1988); Letsinger et al, Nucleoside & Nucleotide13: 1597 (1994); Chapters 2 and 3, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett.4:395 (1994); Jeffs et al, J. Biomolecular NMR 34: 17 (1994);Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, “Carbohydrate Modifications inAntisense Research”, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acidscontaining one or more carbocyclic sugars are also included within thedefinition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995)ppl 69-176). Several nucleic acid analogs are described in Rawls, C & ENews Jun. 2, 1997 page 35. All of these references are hereby expresslyincorporated by reference. These modifications of the ribose-phosphatebackbone may be done to facilitate the addition of ETMs, or to increasethe stability and half-life of such molecules in physiologicalenvironments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention, in general for use ascapture and label probes. In addition, mixtures of naturally occurringnucleic acids and analogs can be made (e.g. in general, the label probescontain a mixture of naturally occurring and synthetic nucleotides).

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acids (particularly in the case of thetarget nucleic acids) may be DNA, both genomic and cDNA, RNA or ahybrid, where the nucleic acid contains any combination of deoxyribo-and ribonucleotides, and any combination of bases, including uracil,adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine,isocytosine, isoguanine, etc. One embodiment utilizes isocytosine andisoguanine in nucleic acids designed to be complementary to otherprobes, rather than target sequences, as this reduces non-specifichybridization, as is generally described in U.S. Pat. No. 5,681,702,disclosure of which is hereby incorporated by reference. As used herein,the term “nucleoside” includes nucleotides as well as nucleoside andnucleotide analogs, and modified nucleosides such as amino modifiednucleosides. In addition, “nucleoside” includes non-naturally occurringanalog structures. Thus for example the individual units of a peptidenucleic acid, each containing a base, are referred to herein as anucleoside.

As will be appreciated by those in the art, a large number of analytesmay be detected using the present methods; basically, any target analytefor which a binding ligand, described below, may be made may be detectedusing the methods of the invention.

Thus, the systems of the invention are used in assays of target analytesthat then allow the diagnosis, prognosis or treatment options of diseasebased on the presence or absence of the target analytes. For example,the systems of the invention find use in the diagnosis orcharacterization of pathogen infection (including bacteria (both grampositive and gram negative bacteria, and/or the ability to distinguishbetween them), viruses (including the presence or absence of viralnucleic acid as well as the isotypes of the virus, for example in thecase of hepatitis C virus (HCV) or respiratory viruses), fungalinfection, antibiotic drug resistance, genetic diseases (includingcystic fibrosis, sickle cell anemia, etc.). Included in the definitionof genetic disease for the purposes of this invention are geneticconditions that do not necessarily cause disease but can result in analternative treatment options. For example, single nucleotidepolymorphisms (SNPs) in many cytochrome p450 enzymes cause differenttherapeutic drug processing, such as in the case of warfarin testing,where a patient may be diagnosed as a “slow”, “normal” or “fast”processor, leading to different dosage regimes, or where a drug may becontraindicated for a particular patient based on the patient'sgenetics, or where selection between two or more drugs is aided by theknowledge of patient's genetics.

Multiplex Cartridge

A multiplex cartridge embodying aspects of the present invention isshown in FIGS. 1-4. As shown in FIG. 4, the multiplex cartridgecomprises an assembly that includes a sample preparation module 70. Thesample preparation module 70 includes various wells, inlet and outletports, fluid channels, mixing mechanisms, valves, and other componentsfor receiving, transporting, intermingling, mixing, and performing otherprocesses on fluid sample materials and process fluids, such as reagentsand buffers, in a manner that will be described in further detail below.The sample preparation module 70 comprises a substrate 72, with a topseal 56 secured to a top surface thereof and a bottom seal 230 securedto a bottom surface thereof. The substrate 72 includes a number ofgrooves or open channels formed on the top and bottom surfaces thereof.Each of the grooves may connect to one or more inlet ports comprising ablind hole formed in the top surface of the substrate 72 and/or to oneor more outlet ports comprising a blind hole formed in the bottomsurface of the substrate 72. The top seal 56 and bottom seal 230 coverthe top and bottom, respectively, of the substrate 72 and havingopenings that align with the inlets and outlets formed in the substrate72, thereby forming a network of conduits, or enclosed channels, throughwhich a fluid—e.g., liquid, gas, solution, emulsion, liquid-solidsuspension, etc.—may flow from one part of the sample preparation module70 to another and inlet ports and outlet ports through which fluids mayflow into and out of, respectively, the sample preparation module 70. Invarious embodiments, the sample preparation module 70 is transparent ortranslucent and is made from, for example, polycarbonate, polypropylene,acrylic, Mylar, acrylonitrile butadiene styrene (“ABS”), or othersuitable polymers

A rotary mixer 192 is operatively disposed within a mixing well 90(described below) formed in the substrate 72. In various embodiments,the rotary mixer 192 can be used, for example, to grind up solidsamples, maximize exposure of sample to capture beads, mix sample withchemical lysis buffer, mix magnetic beads with binding buffer (typicallymagnetic beads cannot be stored in their binding buffer and thus must becombined only at the time of use), etc.

A sample cap 84 is provided to enclose a sample well 78 (describedbelow) formed in the substrate 72. A plurality of deformablecompartments (or blisters) 34 a, 36 a, 38 a, 40 a, 42 a, and 44 aresupported on top of the substrate sample preparation module 70. Eachdeformable compartment may contain a fluid and may be connected to afluid channel within the sample preparation module 70, via one of theinlet ports, by an openable connection that is initially closed toprevent fluid from flowing from the blister into the channel. Uponapplication of a compressive force to the exterior of the blister,increased pressure within the blister ruptures or otherwise opens oralters the openable connection to permit fluid flow from the blisterinto an associated inlet port and channel of the sample preparationmodule 70.

An upper shroud 12 is disposed over a top portion of the cartridge abovethe sample preparation module 70 and includes openings corresponding innumber, size, and shape to the various deformable compartments supportedon the sample preparation module 70. As can be appreciated from FIG. 1,the deformable compartments are recessed within the openings formed inthe upper shroud 12, thereby providing some protection for thedeformable compartments while allowing each compartment to be compressedfrom above by an actuator. In various embodiments, the upper shroud 12further includes an inlet optical port 14 and an outlet optical port 16to enable monitoring of fluid movement through a particular portion ofthe sample preparation module 70, as will be described in further detailbelow. The upper shroud 12 may further include a label panel 24 on whichidentifying information may be placed, such as, human and/ormachine-readable indicia (e.g., a barcode).

The upper shroud 12 may further include valve actuator tabs, such as asample valve actuator tab 18 and a waste valve actuator tab 20. Thevalve actuator tabs 18 and 20 are resilient, flexible tabs formed in theshroud that will deflect upon application of an external compressiveforce onto the tab. Each tab further includes a downwardly-extendingactuator post—see, e.g., actuator post 26 in FIG. 1—to thereby actuatean active valve within the sample preparation module 70 and locatedbelow the respective tab 18 or 20, as will be described in furtherdetail below.

Referring to FIG. 4, a reaction module 240 is disposed below the sampleprocessing module 70 and, in various embodiments, may be configured toreceive a processed sample from the sample processing module 70. Invarious embodiments, the reaction module 240 includes process fluidcompartments (containing, for example, reagents, buffers, etc.), meansfor moving fluid droplets in a specified directed manner throughout themodule, means for incubating reaction mixtures, and means for detectingtarget analytes (e.g., nucleic acids),

The reaction module 240 may be secured to the bottom of the samplepreparation module 70 by means of an adhesive gasket 232 that preferablyprovides a fluid-tight seal between the reaction module 240 and thesample preparation module 70. In various embodiments, the reactionmodule 240 comprises a top plate 241 and a bottom, a fluidic processingpanel 354 secured to the bottom of the top plate 241 and which togetherdefine a gap between the bottom surface of the top plate 241 and a topsurface of the fluidic processing panel 354. This gap defines fluidprocessing and reaction spaces within which various steps of the assayor other process are performed.

A lower shroud 30 partially encloses a bottom portion of the cartridgeassembly and cooperates with the upper shroud 12 to define a relativelyhard and ridged outer shell for the cartridge 10. The upper and lowershrouds may provide the cartridge 10 with an asymmetric shape so as toensure that the cartridge 10 is inserted into a processing instrument inonly one orientation. In the illustrated embodiment, the lower shroud 30has rounded edges 32 whereas the upper shroud 12 has relatively squareedges. Thus, a receiving slot of a processing instrument configured toreceive the multiplex cartridge 10 and having a shape conforming to thatof the shroud will ensure that the shroud is always inserted right sideup into the instrument. In addition, the lower shroud 30 may includecontour features, such as longitudinal side grooves 22 that extend onlypartially along the length of the lower shroud 30. Such groovescooperate with corresponding features in a receiving slot of aprocessing instrument to ensure that the cartridge is inserted into theinstrument in the proper direction.

Deformable Fluid Compartments (Blisters)

In general, the blisters are made of a deformable material thatpreferably collapses upon the application of suitable pressure; that is,the materials used to form blisters do not return to their startingshape when the pressure is removed, as this could cause backflow of theapplied reagents. In addition, the blisters may be used once (a singleapplication of pressure is done during the assay) or a number of times(e.g. multiple aliquots of reagent are delivered to either a singlelocation or multiple locations during the assay run). Each blister maycontain a unique process material (e.g., buffer, reagent, immiscibleliquid, etc.), or two or more blisters may contain the same processmaterial. This redundancy may be used to deliver the same processmaterial to multiple locations in the rest of the disposable.

Although the size, number, arrangement, and contents of the compartmentsis largely dictated by the assay or other process that is intended to beperformed in the multiplex cartridge 10, the illustrated embodimentincludes six deformable fluid compartments, or blisters: 34 a, 36 a, 38a, 40 a, 42 a, and 44. A deformable blister may have an associated lanceblister. In the illustrated embodiment, each of deformable fluidblisters 34 a, 36 a, 38 a, 40 a, and 42 a has an associated deformablelance cartridge, or lance blister, 34 b, 36 b, 38 b, 40 b, and 42 b.

Operation of an embodiment of a deformable compartment is described withreference to FIG. 5, which shows a cross section of the deformablecompartment 34 a. In various embodiments, the deformable compartments ofthe multiplex cartridge 10 incorporate features described incommonly-owned U.S. patent application Ser. No. 14/206,867 entitled“Devices and Methods for Manipulating Deformable Fluid Vessels” thecontents of which are hereby incorporated by reference.

When compressing a deformable compartment to displace the fluid contentsthereof, sufficient compressive force must be applied to the blister tobreak, or otherwise open, a breakable seal that is holding the fluidwithin the compartment. The amount of force required to break the sealand displace the fluid contents of a compartment typically increases asthe volume of the compartment increases. To limit the amount ofcompressive force that must be applied to a deformable compartment orblister to break or otherwise open a breakable seal that is holding thefluid within the compartment, a lance blister 34 b is provided inassociation with the deformable compartment 34 a. The deformablecompartment 34 a and the lance blister 34 b may be connected by means ofa channel, which may be initially blocked by a breakable seal. The lanceblister 34 b contains an opening device, e.g., a bead 46 (such as asteel ball bearing), enclosed within the lance blister 34 b andsupported above a fluid port 136 formed in the sample preparation module70 by means of a breakable foil partition, or septum, that retains thebead 46 and the fluid contents within the lance blister 34 b and thedeformable compartment 34 a. Thus, to open the deformable compartment 34a, a compressive force is first applied externally to the lance blister34 b to compress the lance blister 34 b and force the bead 46 throughthe foil partition blocking the fluid port 136. After the fluid port 136is opened, the fluid contents of the deformable compartment 34 a can bedispensed into the fluid port 136 relatively easily by application of anexternal compressive force to the deformable compartment 34 a. Theamount of pressure required to compress the lance blister 34 b and forcethe bead 46 through the foil partition is much less than that requiredto compress the primary compartment 34 a and create sufficient pressureto open a burstable seal. Fluid flowing into the fluid port 136 willnext flow through a horizontal channel 137, defined by a groove formedin a bottom surface of the substrate 72 and covered by the bottom seal230, to a vertical channel transition 139 and from there to one or moreother points within the sample preparation module 70.

Sample Preparation Module

Various details of a sample preparation module 70 are shown in FIGS.6-15.

The sample well 78 is configured to receive a fluid sample material thatis to be assayed or otherwise processed in the multiplex cartridge 10.As shown in FIGS. 6 and 7, the sample well 78 may be defined by anupright peripheral wall 79 (which is circular in the illustratedembodiment) and a bottom wall, or floor 81. The sample well 78 furtherincludes an inlet snorkel 80 extending up along the peripheral wall 79of the sample well 78 and terminating at a position below the top of theperipheral wall. An exit port 82 is provided in the floor 81 of the well78, and the floor 81 is preferably conical so as to taper downwardtoward the exit port 82.

The sample cap 84 may be provided for closing the sample well 78 after asample material has been deposited into the sample well 78. In oneembodiment, the sample cap 84 comprises a circular cover with an outerperipheral wall that fits over the upright peripheral wall 79 of thesample well 78. The sample cap 84 may include a pivot post 86 defined byradially-resilient locking tabs extending through an opening in thesubstrate 72 and permitting the cap 84 to be pivoted about an axisdefined by the pivot post 86 relative to the sample well 78. After asample material is deposited into the sample well 78, the sample cap 84may be pivoted over the top of the sample well 78 and pushed down overthe sample well 78. A clip, or other detent, 88, extending upwardly maybe provided to catch on and securely lock the sample cap 84 when pusheddown into the clip 88 and to also provide a tactile confirmation thatcap 84 has been securely closed. In some embodiments, the sample cap 84may have a bottom surface that tapers downwardly when the sample cap 84is placed over the sample well 78 (not shown). The conical configurationhelps to reduce the amount of fluid condensate retained on the insidesurface of the sample cap 84 during sample processing in the sample well78.

The sample preparation module 70 also includes a mixing well 90 formedin the substrate 72. As shown in FIGS. 8A and 9A, the mixing well 90 maybe defined by an upright peripheral wall 91 (which is circular in theillustrated embodiment) and a bottom wall, or floor 93. In variousembodiments, a fluid inlet snorkel 92 extends up the peripheral wall 91of the mixing well 90 and terminates below the top of the wall 91. Invarious embodiments, a pressure snorkel 94 extends up another portion ofthe peripheral wall 91 of the mixing well 90 and terminates at aposition below the top of the wall 91. An exit port 96 allows fluid toexit the mixing well 90 and may comprise a plurality of openings locatednear the center of a downwardly tapered portion of the floor 93 of thewell 90 and surrounding a spindle seat 98 formed at the bottom center ofthe floor 93.

The rotary mixer 192 is disposed within the mixing well 90 and includesan upper circular disk 194 supported on an upper edge of the peripheralwall 91 of the well 90. Peripheral gear teeth 198 are formed about theperiphery of the disk 194, and a portion of the teeth 198 project froman outer edge of the upper and lower shrouds 12, 30 of the multiplexcartridge 10 so as to be engageable by an external drive mechanism of aprocessing instrument to effect powered rotation of the rotary mixer192. An O ring 196 is disposed within a peripheral O ring groove aboutthe upper disk 194 below the peripheral gear teeth 198. The O ring 196provides a seal between the rotary mixer 192 and the peripheral wall 91of the well 90. A spindle 200 extends downwardly from the upper disk 194and is seated within the center spindle seat 98 of the mixing well 90. Aplurality of impeller blades 202 extend radially from the spindle 200.

An alternate embodiment of a mixing well 90′ is shown in FIGS. 8B and8C. As shown, mixing well 90′ may be defined by an upright peripheralwall 91′ (which is circular in the illustrated embodiment) and a bottomwall, or floor 93′. A fluid inlet snorkel 92′ extends up an outersurface of the peripheral wall 91′ of the mixing well 90′ and includesan opening 92 a below the top of the wall 91′. A pressure snorkel 94′extends up outer surface of the peripheral wall 91′ of the mixing well90′ and includes an opening 94 a below the top of the wall 91′. An exitport 96′ allows fluid to exit the mixing well 90′ and may comprise aplurality of openings located near the center of a downwardly taperedportion of the floor 93′ of the well 90′ and surrounding a spindle seat98′ formed at the bottom center of the floor 93′. The exit port 96′ andspindle seat 98′ may be substantially identical to the exit port 96 andspindle seat 98, respectively, of the mixing well 90.

With the alternate mixing well 90′ of FIGS. 8B and 8C, a rotary mixerdisposed within the mixing well 90′ may be configured with impellerblades extending radially from a spindle of the mixer substantially tothe inner surface of the peripheral wall 91′. This is opposed to theconfiguration of the rotary mixer 192 configured for operation in themixing well 90, in which the radial impeller blades 202 cannot extendsubstantially to the inner surface of the peripheral wall 91 so as toprovide clearance for the snorkels 92, 94 formed on the inner surface ofthe peripheral wall 91. Having a mixer with impeller blades extending tothe inner surface of the peripheral wall 91′ may, in some circumstances,provide more complete and/or efficient mixing of the contents of themixing well 90′.

Referring to FIG. 9B, the rotary mixer 192′ disposed within the mixingwell 90′ includes an upper circular disk 194′, peripheral gear teeth198′, and an O ring 196′ that may be substantially identical to thecircular disk 194, peripheral gear teeth 198, and an O ring 196 of therotary mixer 192 shown in FIG. 9A. A spindle 200′ extends downwardlyfrom the upper disk 194′. Two or more impeller blades 202′ extendradially from the spindle 200′. The impeller blades 202′ extendsubstantially to the inner surface of the peripheral wall 91′. Invarious embodiments the impeller blades 202′ may be skewed with respectto the spindle 200′ and may further include openings 203 formed thereinto improve the mixing efficiency of the rotary mixer 192′.

Referring again to FIG. 15, which shows a top plan view of the samplepreparation module 70, the sample preparation module 70 may includealignment holes 74 and 76, or other alignment features may be providedin the sample preparation module 70, or some other portion of themultiplex cartridge 10 to facilitate alignment of the multiplexcartridge 10 with a processing instrument, for example, by means of apin or other structure within the instrument extending into eachalignment hole.

The sample preparation module 70 includes a first inlet port 136 formedin a top surface of the module by which a process fluid from thedeformable compartment 34 a may be introduced into the samplepreparation module 70. In one embodiment, the deformable compartment 34a contains a lysis buffer, such as water for hypotonic lysis, or acommercially available lysis buffer, such as those containing chiatropicsalts such as guanidinium salts, and or high/low pH, and/or surfactantssuch as sodium dodecyl sulfate (SDS), TWEEN® 20 (polysorbate 20),TRITON™ X-100 (polyoxyethylene octyl phenyl ether), etc. In some cases,the lysis buffer optionally comprises reagents to disrupt undesiredenzymatic activity, such as DNase and RNase activity, which are thenremoved during the bead capture/elution process (although these can beseparate reagents, either dried or liquid, that can be added as neededdepending on the target analytes and the assay).

After cells of the sample material are lysed, it is often desirable toperform an at least partial purification, to remove other cellular andsample debris from the sample to facilitate the downstream handling andprocessing. Research samples in buffer do not necessarily requirepurification, but even there purification is typically performed. Awell-known technique relies on the use of target capture beads (e.g.,magnetic capture beads) that capture and immobilize the desired targetanalyte(s) away from the cellular and sample debris. In variousimplementations, capture beads and binding buffer are mixed with thesample in lysis buffer after the cells or viruses are disrupted bymechanical and/or chemical means. The capture beads may be magnetic tofacilitate subsequent immobilization of the beads and the target analytebound thereto by selective application of magnetic forces, although aswill be appreciated by those in the art, other implementations mayemploy non-magnetic beads, such as polystyrene or silica beads (forexample, beads may be captured in a zone by size or on an affinitycolumn).

Thus, in various embodiments, the sample preparation module 70 includesa second inlet port 138 by which a process fluid from the deformablecompartment 36 a may be introduced into the sample preparation module70. In one embodiment, the deformable compartment 36 a contains abinding buffer to facilitate the binding of target capture beads, suchas magnetic beads, to one or more target analytes of interest.

In various embodiments, the sample preparation module 70 includes athird inlet port 140 by which a process material from the deformablecompartment 44 may be introduced into the sample preparation module 70.In one embodiment, the deformable compartment 44 contains target capturebeads which may comprise magnetic particles, which, in combination witha binding buffer from the deformable compartment 36 a, binds to ananalyte or analytes of interests within the sample material to therebyisolate and enable the magnetic separation of the analyte(s) of interestfrom the remainder of the sample material.

The capture beads may be coated with a material that facilitates captureof the target analyte(s). For example, for the capture of nucleic acids,the beads can be coated with a negatively charged coating to facilitatethe adsorption of positively charged nucleic acids to the surface, whichare then washed with buffer and then treated with elution buffer toremove the purified nucleic acids from the beads for further processing.As will be appreciated by those in the art, there are a number ofsuitable, commercially available bead systems, including, for example,MagaZorb® Beads from Promega, MagMax from Life Tech, or beads fromQiagen, MoBio, BioRad, etc.

Thus, the target capture beads that may be contained in the deformablecompartment 44 facilitate the purification of the desired target analytewith fluid access to a binding buffer, such as the bind buffer that maybe contained in the deformable compartment 36 a, used in conjunctionwith the capture beads.

In an alternate embodiment, target capture beads may be provideddirectly within the sample preparation module 70, for example, in theform of a lyophilized pellet placed into the mixing well 90 duringassembly of the multiplex cartridge 10 and stored in the mixing well inpellet form until reconstituted by a fluid added to the mixing well 90during use of the multiplex cartridge 10. In this alternate embodiment,the deformable blister 44 may be omitted.

In alternate implementations, capture beads may be functionalized withcapture nucleic acid probes in order to either specifically ornon-specifically pull out nucleic acids. For example, the beads may befunctionalized with random 6-mers, to generally pull out nucleic acids,or with capture probes specific to the desired target nucleic acids. Insome cases, for example when mRNA is the target, beads coated withpoly-T capture probes can be used.

In various embodiments, the sample preparation module 70 furtherincludes a fourth inlet port 142 by which process material from thedeformable compartment 38 a may be introduced into the samplepreparation module 70. In one embodiment, the deformable compartment 38a contains an immiscible fluid (e.g., an oil, such as mineral oil,silicone oil, etc., as discussed in detail below).

In various embodiments, the sample preparation module 70 furtherincludes a fifth inlet port 144 by which a process material from thedeformable compartment 40 a may be introduced into the substrate 72. Inone embodiment, the deformable compartment 40 a contains an elutionbuffer.

In various embodiments, the sample preparation module 70 furtherincludes a sixth inlet port 146 by which process material from thedeformable compartment 42 a may be introduced into the samplepreparation module 70. In one embodiment, the deformable compartment 42a contains a wash buffer.

In various embodiments, the sample preparation module 70 includes afirst outlet port 182, a second outlet port 188, and a third outlet port190 formed in a bottom surface of the sample preparation module 70 bywhich fluid can exit the module 70 and flow into the reaction module240.

It should be noted here that the designation of inlet ports or outletports as the first, second, third, fourth, fifth, or sixth ports ismerely to provide a convenient means for distinguishing one port fromanother and is not meant to be limiting, such as, for example, byspecifying a particular order or sequence by which the ports may beused.

A first fluid channel 150 extends from the first inlet port 136 to thesample well 78. In the diagrams, the fluid channels are represented byparallel lines extending from point to point across the samplepreparation module 70. Each channel may include one or more channeltransition points, represented by a circle in the channel, one of whichis indicated by reference number 151. The channel transition pointrepresents a vertically extending section of channel extending up, froma channel section formed on the bottom of the substrate 72 to a channelsection formed on the top of the substrate 72, or down, from a channelsection formed on the top of the substrate 72 to a channel sectionformed on the bottom of the substrate 72, so that the channel may passover or under another channel within the substrate 72.

A second fluid channel 152 extends from the sample well 78 to the lysischamber inlet 122. A third fluid channel 156 extends from the lysischamber outlet 124 to a fifth fluid channel 162 that extends from thethird inlet port 140 to the mixing well inlet snorkel 92. A fourth fluidchannel 160 extends from the second inlet port 138 to the third inletport 140. A sixth fluid channel 164 extends from the fourth inlet port142 to the first outlet port 182. A seventh fluid channel 166 extendsfrom the fifth inlet port 144 to the second outlet port 188. An eighthfluid channel 168 extends from the mixing well exit port 96 to a passivevalve assembly 220 (described below). A ninth fluid channel 170 extendsfrom a passive valve cavity of the passive valve assembly 220 to acapture compartment 100. A tenth fluid channel 172 extends from anactive valve assembly 204 to an active valve assembly 219. An eleventhfluid channel 174 extends from the active valve assembly 219 to a wastechamber 102. A twelfth fluid channel 176 extends from the sixth inletport 146 to the capture compartment 100. A thirteenth fluid channel 178extends from the capture compartment 100 to the active valve assembly204. A fourteenth fluid channel 180 extends from the active valveassembly 204 to the third outlet 190.

It should be noted here that the designation of the various fluidchannels as the first, second, third, fourth, fifth, etc. fluid channelsis merely to provide a convenient means for distinguishing one port fromanother and is not meant to be limiting, such as, for example, byspecifying a particular order or sequence in which the fluid channelsmay be used or a particular direction in which fluids flow through thechannels.

In various embodiments, the sample preparation module 70 furtherincludes a passive valve assembly 220 adjacent the mixing well 90. Inone embodiment, the passive valve assembly 220 is configured such thatthe passive valve assembly 220 is closed if pressure within the mixingwell 90 is below a threshold pressure and thus fluid within the mixingwell 90 is retained. On the other hand, if pressure is allowed toincrease within the mixing well 90, at a sufficient pressure level,above the threshold pressure, the passive valve assembly 220 will beopened, thereby permitting fluid within the mixing well to escape viathe exit port 96 and the eighth fluid channel 168 connecting the mixingwell exit port 96 to the passive valve assembly 220.

Details of the passive valve assembly 220 are shown in FIGS. 10 and 11.The valve assembly 220 comprises a valve cavity 222 formed in thesubstrate 72 and an inlet 224 formed in the substrate 72 and extendingupwardly into the valve cavity 222. A valve 229, which may comprise aBelleville valve, is disposed within the valve cavity 222 over the inlet224. A retainer 226 is disposed over the valve 229. An outlet 228extends radially from the valve cavity 222.

In an unpressurized condition, the valve 229 and the retainer 226 are atrest at the bottom of the valve cavity 222, with the valve 229 coveringthe inlet 224. The retainer 226 may be biased in a down position, e.g.,by a suitable spring or the like. Accordingly, fluid flowing from theinlet 224 is not able to pass into and through the valve cavity 222, andthus, fluid is not able to escape the mixing well 90. On the other hand,if fluid in the inlet 224 is sufficiently pressurized to overcome anyforce (e.g., spring bias) holding the retainer 226 in a down position(e.g., about 3 to 5 psi), the valve 229 and the retainer 226 will belifted off the bottom of the valve cavity 222 thereby opening the inlet224 and allowing fluid to flow into the valve cavity 222 and out of theoutlet 228.

The sample preparation module 70 may further include a pump port 104 bywhich an external source of pressure may be coupled to the samplepreparation module 70. The pump port 104 is connected, via a pressureconduit 106 to the sample well 78 so that pressure applied at the pumpport 104 will pressurize the sample well 78 to motivate the contents ofthe sample well 78 out of the well.

The sample preparation module 70 may further include a passive valveport 108 is connected, via a valve conduit 110 to the pressure snorkel94 of the mixing well 90. If the passive valve port 108 is open,pressure will not build up within the mixing well 90, and the passivevalve assembly 220 will remain closed. If the passive valve port 108 isclosed, pressure will build up within the mixing well 90 and the passivevalve assembly 220 will open so that the contents of the mixing well 90can flow from the well.

Some organisms, such as viruses and many bacteria, can be lysedchemically by the addition of a lysis buffer with or without elevatedtemperature or proteolytic enzymes. Some organisms are difficult to lyseby chemical and/or enzymatic methods and require mechanical disruptionor shearing of the cell membranes. As such, an optional component of themultiplex cartridge 10 is an impeller component, wherein the impeller isactivated to grind or break up solid components such that individualcells are more accessible to lysis buffer and so that more targetanalytes are released. The impeller imparts turbulent action to thefluid in which lysis beads are contained. The primary lysis action isdue to bead collisions with target organisms, which are thereby lysed,breaking them open and exposing the target nucleic acids. The presenceof the lysis buffer inhibits the DNases or RNases which may destroy theRNA or DNA targets once the cells are disrupted. In various embodiments,the impeller is like a paddle wheel that rotates very fast.

Thus, in various embodiments, the sample preparation module 70 furtherincludes a lysis chamber 120 with a driven agitator, such as a motorizedbead mixer mechanism, disposed therein. The driven agitator is disposedat least partially within the lysis chamber 120 and is constructed andarranged to agitate fluid flowing through the processing chamber. Thefluid flowing through the lysis chamber may comprise a mixture of samplematerial, lysis buffer, and lysis beads. The lysis beads may comprisesilica (ceramic) beads (of, e.g., 100 μm diameter) that are dispensedinto the lysis chamber 120 during assembly of the multiplex cartridge10. The bead mixer comprises a motor 128 with an impeller 130 mounted onan output shaft of the motor (see FIG. 2). Fluid flows into the lysischamber 120 through an inlet 122 and flows out of the lysis chamber 120through an outlet 124. A mesh filter may be provided in front of theinlet 122 and/or the outlet 124. The mesh filter(s) have a pore sizeconfigured to retain the lysis beads within the lysis chamber 120 whileallowing sample fluid to flow into and out of the lysis chamber 120. Inoperation, the motor 128 rotates the impeller 130 at a high rate ofrotation (e.g., about 5,000 to about 100,000 rpm, preferably about10,000 to about 50,000 rpm, more preferably about 20,000 to about 30,000rpm), so that fluid within the lysis chamber 120, which may includesample material and lysis beads, is vigorously agitated by the rotatingimpellor, thereby assisting the lysis beads in disrupting the molecularstructure of the sample material. Thus, the sample mixture flowing outof the lysis chamber 120 is more completely lysed than it would bewithout the bead mixer.

A suitable motor 128 of the bead mixer includes Feiying, ModelFY0610-Q-04170Y from Jinlong Machinery. The motor may be powered by atemporary connection of the multiplex cartridge 10 to an external powersource of an instrument in which the cartridge 10 is being processed.Control of the motor 128 may be implemented by means of logic elementsprovided externally and/or internally of the cartridge 10. In oneembodiment, a mixer printed circuit board (“PCB”) is provided within thelower shroud 30 that controls operation of the bead mixer motor 128. Themixer motor 128 is ideally only operated when fluid is flowing throughthe lysis chamber 120. Fluid flowing into the lysis chamber 120 can bedetected by an optical sensor through the inlet optical port 14 formedin the upper shroud 12 (see FIG. 2), which is aligned with an inletoptical sensing chamber 154 (see, e.g., FIG. 15), so that the bead mixermotor 128 can be activated, for example, upon detection of the forwardend of a fluid stream flowing through the inlet optical sensing chamber154 toward the lysis chamber 120. Similarly, fluid flowing out of thelysis chamber 120 can be detected by an optical sensor through theoutlet optical port 16 (see FIG. 2), which is aligned with the outletoptical sensing chamber 158 (see FIG. 15), so that the bead mixer motor128 can be deactivated, for example, upon detection of the trailing endof a fluid stream flowing through the outlet optical sensing chamber158.

The sample preparation module 70 further includes two active valveassemblies 204, 219. The valve assembly 204 is known as the sample valveassembly and is positioned at the junction of the tenth fluid channel172, the thirteenth fluid channel 178, and the fourteenth fluid channel180 and controls flow from the thirteenth fluid channel 178 into thefourteenth fluid channel 180. Valve assembly 219 is known as the wastevalve assembly and is positioned at the junction of the tenth fluidchannel 172 and the eleventh fluid channel 174 and controls flow fromthe tenth fluid channel 172 to the eleventh fluid channel 174 and thewaste chamber 102.

Details of an active valve assembly, e.g., the valve assembly 204, areshown in FIGS. 13 and 14. The valve assembly 204 comprises a valvecavity 210 formed in the substrate 72. An inlet conduit 208 leads intothe valve cavity 210, and an outlet channel 212 extends out of thecavity 210. An access opening 206 is formed in the top seal 56 disposedatop the substrate 72. A flexible valve membrane 216 is secured to anunderside of the top seal 56 beneath the access opening 206 by means ofan adhesive 214 surrounding the access opening 206. In the undeflected,or unactuated, position, as shown in FIG. 13, fluid may flow into thevalve cavity 210 through the inlet 208 and flow out of the valve cavity210 through the outlet 212. Accordingly, fluid flow through the valveassembly 204 is unimpeded. As shown in FIG. 14, when an external valveactuator 218 presses down through the access opening 206 to deflect thevalve membrane 216 over the outlet 212, fluid flow through the valveassembly 204 is blocked. The valve actuator 218 may comprise an actuatorpost 26 of the actuator tab 20 formed in the upper shroud 12 (see FIG.1). Specifically, valve actuator tab 18 is aligned with the active valveassembly 204, and valve actuator tab 20 is aligned with the active valveassembly 219.

In various embodiments, the sample preparation module 70 furtherincludes a waste chamber 102 (or more than one waste chamber) configuredto receive and container excess or used fluids.

Reaction Module—Top Plate

Details of the reaction module 240, and the top plate 241 in particular,are shown in FIGS. 24-31. Referring to FIGS. 24 and 26, which show a topperspective view and a top plan view, respectively, of the top plate241, the top plate 241 includes an upper perimeter wall 256 projectingabove a top surface 242 of the top plate 241 and at least partiallycircumscribing the top surface 240 at a location offset inwardly fromthe outer edges of the top plate 241. The upper perimeter wall 256 has acontinuous open channel or groove 258 formed along its top edge whichprovides a seat for the adhesive gasket 232 securing the reaction module240 to the sample preparation module 70. See FIG. 4. The upper perimeterwall 256 forms a recessed area 260 surrounded by the upper perimeterwall 256 on the top surface 242. See also FIGS. 28 and 29.

Top plate 241 can take on a number of configurations and can be made ofa variety of materials. Suitable materials include, but are not limitedto, fiberglass, TEFLON®, ceramics, glass, silicon, mica, plastic(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polycarbonate,polyurethanes, and derivatives thereof, etc.), etc. A particularlypreferred top plate material is polycarbonate.

An alignment fork 246 extends from one end of the top plate 241, and analignment loop 244 extends from an opposite end of the top plate 241.The alignment fork 246 and alignment loop 244 are configured to receivealignment pins in an instrument for processing the multiplex cartridge10 to ensure proper alignment of the cartridge 10, as described in moredetail below.

The top plate 241 further includes a sample compartment 266 with aninlet port 268 that is in fluid communication with the third outlet port190 of the sample preparation module 70.

The top plate 241 further includes a rehydration (elution) buffercompartment 276 having an inlet port 278 that is in fluid communicationwith the second outlet port 188 of the sample preparation module 70. Adetection buffer compartment 280 contains an initially-dried detectionbuffer (applied to a portion of the top plate 241 forming the detectionbuffer compartment 280 or a portion of the fluidic processing panel 354covering the detection buffer compartment 280) that is reconstitutedwith an amount of the reconstitution buffer dispensed into therehydration buffer compartment 276 and transferred to the detectionbuffer compartment 280. In one embodiment, the detection buffercompartment 280 has a capacity of 120-160 μl. In various embodiments,top plate 241 includes a connecting passage 274 between the detectionbuffer compartment 280 and the rehydration buffer compartment 276. Thedetection buffer compartment 280 may further include a port 282 forinjecting a buffer into the compartment 280 during a manufacturingprocess and/or for venting the compartment 280.

FIGS. 25 and 27 show a bottom perspective view and a bottom plan view,respectively, of the top plate 241. Referring to FIGS. 25 and 27, inaddition to FIGS. 24 and 26, the top plate 241 further includes a buffercompartment 296, which, in one embodiment, contains a PCR buffer/enzymein a dried form (applied to a portion of the top plate 241 forming thebuffer compartment 296 or to a portion of the fluidic processing panel354 (see FIGS. 4 and 58) covering the buffer compartment 296), to belater reconstituted (rehydrated) by an amount of rehydration buffer fromthe rehydration buffer compartment 276. In one embodiment, the buffercompartment 296 has a capacity of about 20 μl. A port 298 is providedfor injecting the PCR buffer/enzyme into the compartment during themanufacturing process and/or for venting the buffer compartment 296.

The top plate 241 further includes a second buffer compartment 300 whichmay contain an exonuclease reagent in a dried form (applied to a portionof the top plate 241 forming the second buffer compartment 300 or to aportion of the fluidic processing panel 354 covering the second buffercompartment 300), to be later reconstituted by an amount of rehydrationbuffer from the rehydration buffer compartment 276. In one embodiment,the second buffer compartment 300 has a capacity of about 20 μl. A port302 may be provided for injecting buffer into the second buffercompartment 300 during a manufacturing process and/or for venting thecompartment 300. A weir 306 may be provided between the rehydrationbuffer compartment 276 and the second buffer compartment 300 to permitfluid flow from the rehydration buffer compartment 276 into thecompartment 300.

The top plate 241 further includes a lower perimeter wall 290circumscribing the bottom of the top plate 241. The lower perimeter wall290 defines a recess surrounded by the perimeter wall 290 configured toreceive a panel, such as the fluidic processing panel 354, to enclosethe lower half of the top plate 241. A raised panel support 290surrounds the outer periphery of the lower surface of the top plate 241just inside the perimeter wall 290. Area 294 inside the panel support292 is slightly recessed with respect to the panel support 292, so thata panel inserted within the perimeter wall 290 is supported on the panelsupport surface 292, and the recess 294 defines a gap 295 (see FIGS. 28,29) between the panel and the top plate 241.

The top plate 241 may further include fluid inlet ports 250, 252, atleast one of which is in fluid communication with the first outlet port182 of the sample preparation module 70. The inlet ports 250, 252provide a fluid communication with the gap 295 between the bottomsurface of the reaction top plate 241, e.g., at the area 294, and thefluidic processing panel 354 enclosing the bottom surface of the topplate 241.

The top plate 241 further includes detection compartments 350 a, 350 b,350 c, and 350 d, each with an inlet port or venting port 352. Theillustrated embodiment includes four detection compartments 350 a-d,though one can easily envision alternative configurations of the topplate 241 comprising a smaller or larger number of the detectioncompartments 350.

Area 304 on the lower surface comprises a processing area that isslightly recessed relative to the area 294, thereby forming a larger gapbetween the top plate 241 and a lower panel in the area 304 than in thearea 294.

The reaction module 240 may further include one or more bubble traps 340that are formed in the top plate 241. Each bubble trap 340 includes abubble capture hood 342 formed in the top plate 241 which slopesupwardly toward a vent opening 344. In one embodiment, rising airbubbles generated by fluid movement beneath the bubble trap are capturedin the capture hood 344 and released through the vent opening 344. Thecapture hood may be shaped as to conform to a fluid movement pathbeneath or adjacent to the bubble trap. In the illustrated embodiment,five bubble traps 340 having elongated capture hoods 342 are positionedabove four fluid movement paths, each located below and between twoadjacent bubble traps 340, as will be described in further detail below.

Details of the fluid inlet 252 are shown in FIG. 30. As noted, the fluidinlet 252 may be aligned with first fluid outlet 182 of the samplepreparation module 70. The fluid inlet 252 may have an inwardly tapered,frustoconical shape, wherein the size of the outlet 182 above the inlet252 is narrower than the upper end of the inlet 252. This helps ensurethat fluid dispensed through the outlet 182 is captured by the inlet252.

Details of the sample compartment 266, the rehydration buffercompartment 276, and the detection buffer compartment 280 are shown inFIG. 31. The sample compartment 266 is configured to receive an amount(e.g., 200 μl) of magnetic beads with bound target analyte (e.g., DNA,nucleic acid) from the sample preparation module 270 through the inletport 268. The inlet port 268 of the sample compartment 266 preferablyhas a conical shape and is aligned with the third outlet 190 of thesample preparation module 70. In various embodiments, the third outlet190 passes through a tapered nipple 322 to minimize hanging dropletsfrom the end the outlet 190. The inlet port 268 is also preferablytapered with its widest end at the top to thereby ensure that fluiddispensed through the outlet 190 is captured in the inlet port 268. Theoutlet 190 and the inlet port 268 are configured such that there is asmall gap therebetween. This gap comprises part of an interstitial space308 between the top of the top plate 241 of the reaction module 240 andthe bottom of the sample preparation module 70. This gap provides a trapfor collecting any air bubbles contained in fluids within the reactionmodule 240, especially air bubbles that may be generated when dispensingfluid from the outlet 190 into the inlet port 268.

The rehydration buffer compartment 276 is configured to receive anamount (e.g., 200 μl) of a buffer solution that is suitable forrehydration of dried reagents and elution of nucleic acid from beadsfrom the sample preparation module 270 through the inlet port 278. Theinlet port 278 of the rehydration buffer compartment 276 is aligned withthe second outlet 188 of the sample preparation module 70. Again, theoutlet 188 preferably flows through a tapered nipple 320, the end ofwhich is spaced apart from the inlet port 278, which is also tapered.Again, the space between the end of the nipple 320 and the inlet port278 allows gas bubbles within the fluid flowing between the outlet 188and the inlet port 278 to escape into the interstitial space 308.

Reaction Module—Fluidic Processing Panel

Referring to FIGS. 58, 59, in various embodiments, the reaction module240 of the multiplex cartridge 10 includes a fluidic processing panel354, secured to the bottom of the top plate 241. The fluidic processingpanel 354 is surrounded peripherally by the perimeter wall 290 and issupport on and secured to the panel support 292, for example by an oiland temperature-resistant adhesive. The fluidic processing panel 354facilitates a number of functionalities of the multiplex cartridge 10,such as fluid movement and analyte detection. Such fluid movements mayinclude transporting one or more droplets of fluid along fluid transportpathways, mixing fluids by moving one or more droplets in an oscillatoryfashion (e.g., linearly back and forth or in a continuous (e.g.,circular, oval, rectangular) path), combining fluid droplets that maycontain different materials, splitting droplets into two or more smallerdroplets, etc.

The fluidic processing panel 354 includes a substrate 356. Suitablesubstrates include metal surfaces such as gold, electrodes as definedbelow, glass and modified or functionalized glass, fiberglass, ceramics,mica, plastic (including acrylics, polystyrene and copolymers of styreneand other materials, polypropylene, polyethylene, polybutylene,polyimide, polycarbonate, polyurethanes, TEFLON®, and derivativesthereof, etc.), GETEK® (a blend of polypropylene oxide and fiberglass),etc., polysaccharides, nylon or nitrocellulose, resins, silica orsilica-based materials including silicon and modified silicon, carbon,metals, inorganic glasses and a variety of other polymers, with printedcircuit board (PCB) materials being particularly preferred.

In various embodiments, the fluidic processing panel 354 may dividedinto a number of distinct functional areas or processing zones, whichcan be spatially overlapping or spatially distinct or partiallyspatially separate and partially spatially distinct.

In various embodiments, fluid reaction processing within the reactionmodule 240 is at least partially based on microfluidic fluidmanipulation using so-called electrowetting techniques to formmicrodroplets that can be manipulated both spatially and biochemically.

In general, electrowetting is the modification of the wetting propertiesof a hydrophobic surface (such as PCB) with an applied electric field.In an electrowetting system, the change in the substrate-electrolytecontact angle due to an applied potential difference results in theability to move the electrolyte on a surface. Essentially, as describedin U.S. Pat. No. 6,565,727, the disclosure of which is hereby expresslyincorporated by reference, by applying an electric potential to anelectrode (or group of electrodes) adjacent to a drop of polar liquid(e.g., one containing a target analyte), the surface on these electrodesbecomes more hydrophilic and the drop is pulled by a surface tensiongradient to increase the area overlap with the charged electrodes. Thiscauses the drop to spread on the surface, and, by subsequently removingthe potential or activating different electrodes, the substrate returnsto a hydrophobic state, resulting in the drop moving to a newhydrophilic area on the substrate. In this way, the drops can bephysically and discretely moved on the planar surface of the substrateto different processing zones, for processing, handling, and detection.The drops can be moved at varied speeds, split (e.g. a single drop canbe split into two or more drops), pulsed and/or mixed (two or more dropsmerged onto the same location and then either split or moved as one). Inaddition, electrowetting can instigate mixing within a single droplet.As described in more detail below, drops can also be used to rehydratedry reagents stored at different locations on the PCB substrate. Onetypical characteristic of electrowetting is precise manipulation of verysmall fluid volumes. For example, isolated target nucleic acid can beeluted at a very high concentration in less than 10 μl prior to PCRamplification, compared to 100 μl elution volumes and much lower targetanalyte concentrations featured in other systems. In addition,electrowetting allows fluid paths to be altered in development and inthe product via software, without the need to make any changes to thephysical interface (e.g., new valves, fluid paths, etc.).

Exemplary microfluidic systems utilizing electrowetting techniques aredescribed in U.S. Patent Pub. Nos. 2013/0252262, 2013/0233712,2013/0233425, 2013/0230875, 2013/0225452, 2013/0225450, 2013/0217113,2013/0217103, 2013/0203606, 2013/0178968, 2013/0178374, 2013/0164742,2013/0146461, 2013/0130936, 2013/01 18901, 2013/0059366, 2013/0018611,2013/0017544, 2012/0261264, 2012/0165238, 2012/0132528, 2012/0044299,2012/0018306, 2011/0311980, 2011/0303542, 2011/0209998, 2011/0203930,2011/0186433, 2011/0180571, 2011/01 14490, 2011/0104816, 2011/0104747,2011/0104725, 2011/0097763, 2011/0091989, 2011/0086377, 2011/0076692,2010/0323405, 2010/0307917, 2010/0291578, 2010/0282608, 2010/0279374,2010/0270156, 2010/0236929, 2010/0236928, 2010/0206094, 2010/0194408,2010/0190263, 2010/0130369, 2010/0120130, 2010/0116640, 2010/0087012,2010/0068764, 2010/0048410, 2010/0032293, 2010/0025250, 2009/0304944,2009/0263834, 2009/0155902, 2008/0274513, 2008/0230386, 2007/0275415,2007/0242105, 2007/0241068, U.S. Pat. Nos. 8,541,176, 8,492,168,8,481,125, 8,470,606, 8,460,528, 8,454,905, 8,440,392, 8,426,213,8,394,641, 8,389,297, 8,388,909, 8,364,315, 8,349,276, 8,317,990,8,313,895, 8,313,698, 8,304,253, 8,268,246, 8,208,146, 8,202,686,8,137,917, 8,093,062, 8,088,578, 8,048,628, 8,041,463, 8,007,739,7,998,436, 7,943,030, 7,939,021, 7,919,330, 7,901,947, 7,851,184,7,822,510, 7,816,121, 7,815,871, 7,763,471, 7,727,723, 7,439,014,7,255,780, 6,773,566, and 6,565,727, the respective disclosures of whichare hereby incorporated by reference.

Thus, in various embodiments, the fluidic processing panel 354 comprisesa grid of electrodes which form and define discrete processing zones,including pathways, for fluid droplets as appropriate for the assays orother process(es) being performed in the reaction module 240. Ingeneral, a “spot” or “location” or “pad” (sometimes referred to as an“electrowetting pad” or “EWP”) is generally depicted in the figures as arectangle wherein the lines forming the sides of the rectangle representelectrodes, such that a droplet moves along a path in discrete steps,from pad to pad. By manipulating the electrode grid, the droplets can beselectively moved in any of four directions as needed: forward,backward, left, or right, relative to a current position. Thus, invarious embodiments the fluidic processing panel 354 includes a grid ofetched electrodes forming a network of pads for moving sample dropletsfrom sample preparation through detection of target analytes.

In the illustrated embodiment, the electrodes formed on the substrate356 of the fluidic processing panel 354 define a number of discrete,functional regions that provide for movement and/or collection of fluiddroplets. As shown in FIGS. 26, 27, and 59, these zones include a samplebead zone 368 spatially corresponding to the sample compartment 266 ofthe top plate 241, a hybridization zone 370 spatially corresponding tothe detection buffer compartment 280 of the top plate 241, a rehydrationbuffer zone 372 spatially corresponding to the rehydration (elution)buffer compartment 276 of the top plate 241, an exonuclease reagent zone374 spatially corresponding to the second buffer compartment 300 of thetop plate 241, and a PCR reagent zone 376 spatially corresponding to thebuffer compartment 296 of the top plate 241. Other zones defined on thefluidic processing panel 354 include electrosensor zones 360 a, 360 b,360 c, and 360 d corresponding spatially to detection compartments 350a, 350 b, 350 c, and 350 d, respectively, and which further includeelectrosensor arrays 363 a, 363 b, 363 c, and 363 d, respectively. Stillother pathways defined on the fluidic processing panel 354 includethermocycling, or PCR, pathways 364 a, 364 b, 364 c, and 364 d, eachbeing located spatially below and between two adjacent bubble traps 340of the top plate 241.

Electrodes of the fluidic processing panel 354 may further define anexonuclease zone 384.

Electrodes of the fluidic processing panel 354 may further definedetection mixing zones, which, in the illustrated embodiment comprisefour groups of nine electrode pads indicated by reference numbers 385 a,385 b, 385 c, and 385 d.

The fluidic processing panel may further include a number of connectorpad arrays configured to contact and make electrical connections withconnector pins (e.g., pogo pins) located within the processinginstrument, as will be described in further detail below. Theillustrated embodiment includes seven connector pad arrays: 358 a, 358b, 358 c, 358 d, 358 e, 358 f, and 358 g.

As will be appreciated by those in the art, there are a wide number ofelectrode grid configurations that can be employed in the multiplexcartridge 10, including, without limitation, configurations describedherein. Exemplary electrowetting electrode configurations for differentutilities are shown in previously-incorporated U.S. Pat. No. 8,541,176.

In general, preferred materials for the fluidic processing panel 354include printed circuit board materials. In various embodiments, circuitboard materials are those that comprise an insulating substrate (e.g.,the substrate 356) that is coated with a conducting layer and processedusing lithography techniques, particularly photolithography techniques,to form the patterns of electrodes and interconnects (sometimes referredto in the art as interconnections or leads). The insulating substrate isgenerally, but not always, a polymer. As is known in the art, one or aplurality of layers may be used, to make either “two dimensional” boards(e.g., all electrodes and interconnections in a plane, “edge cardconnectors”) or “three dimensional” boards (wherein the electrodes areon one surface and the interconnects may go through the board to theother side or wherein electrodes are on a plurality of surfaces). Threedimensional systems frequently rely on the use of drilling or etching toform holes, or vias, through the substrate, followed by electroplatingwith a metal such as copper, such that the “through board”interconnections are made. Circuit board materials are often providedwith a foil already attached to the substrate, such as a copper foil,with additional copper added as needed (for example forinterconnections), for example by electroplating. The copper surface maythen need to be roughened, for example through etching, to allowattachment of the adhesion layer.

In one embodiment, electrical connections from both the electrowettingelectrode grids and detection electrodes, i.e., the connector pad arrays360 a-g, extend through the panel to produce a so-called land grid arraythat can interface to a pogo pin or like connector to make connectionsfrom the chip to a processing instrument. In various embodiments, thesurface of the fluidic processing panel 354 (e.g., the PCB with theelectrode grids) is coated with a film of a substance to facilitate theelectrowetting mechanism and clean transport from pad to pad. In variousembodiments, the surface is coated with a polyimide film, such asKAPTON® from DuPont (e.g., black or yellow KAPTON®), which forms adielectric layer. The surface properties of the dielectric layer areimportant to facilitate electrowetting and to attenuate the voltagebeing used in order to prevent electrolysis in the aqueous droplet. Inaddition, the Kapton® or similar surface, such as a solder mask, must becoated with a hydrophobic coating, such as Paralyene, TEFLON®(polytetrafluoroethylene), CYTOP® fluoropolymers, to name a few, torender the surface hydrophobic, which is required for electrowetting tofunction.

As will be appreciated by those in the art, the form of the reagentprovided in the reaction module 240 will depend on the reagent. Somereagents can be dried or in solid form (for example, when particularbuffers are to be used), others can be lyophilized, etc. Particularlyuseful embodiments utilize dried reagents with added stabilizers, suchas salts, sugars, polysaccharides, polymers or proteins such asgelatins, etc. as will be appreciated by those in the art. For example,Biomatrica produces commercial stabilizers for use in the presentsystem.

As will be appreciated by those in the art, if used, the dried reagentscan be rehydrated in one of two general ways. Either liquid from thesample preparation module 70 is introduced at the appropriate pad (orzone) or the sample itself serves as an aqueous solvent to put the solidreagents into solution. For example, the appropriate resuspension buffer(which can be water, in some cases) can be added through the top plate241 from the sample preparation module 70 to a particular pad torehydrate the reagent(s), and then the reagent droplet can be mergedwith the sample droplet.

Alternatively, the drops containing the target analyte (for example, inelution buffer used to liberate the target analytes from the capturebeads) may be transported to a pad containing the dried reagent(s),which are then suspended in the drop itself. One benefit of thisembodiment is that the ultimate volume of a droplet does not increasesignificantly, as it does when a drop of reagent is merged with a dropof sample. This may be particularly useful in situations where multiplereagent additions are required.

The number, type and quantity of the different reagents will depend onsample, the target analyte and the desired reaction. For example, fornucleic acid target sequences in a standard PCR reaction, when thestarting sample is DNA, the on-board dried reagents include RT-PCRbuffer, PCR enzyme (e.g. a Taq polymerase), dNTPs, PCR primers,exonuclease, signal probes, signal buffer and detection buffers (withthe lysis buffer, the binding buffer, the elution buffer, the (optional)reconstitution buffer(s), and magnetic bead suspension all beingcontained in the sample preparation module 70, rather than dried on thefluidic processing panel 354). Exemplary embodiments are outlinedherein. However, as will be appreciated by those in the art, any numberof configurations of dried reagents and liquid reagents in the samplepreparation module 70 can be used.

The compartment within the reactor module 240 formed between the fluidicprocessing panel 354 and top plate 241 described above, is generallyfilled with a fluid in which the target analyte droplets (usuallyaqueous solutions) are immiscible, and this immiscible fluid isgenerally less polar than the solution of the drop. As described in U.S.Pat. No. 8,541,177, the disclosure of which is hereby incorporated byreference, there are two general ways of isolating drops on padsincluding filling the compartment with an immiscible fluid includingimmiscible liquids and immiscible gases, or by using the immiscibleliquid as a droplet encapsulant, for example giving the droplet a shellof oil by passing the droplet through an air/oil interface.

Particularly suitable immiscible fluids for use in the nucleic aciddetection assays described herein include, but are not limited to,silicone oils, mineral oil, fluorosilicone oils; hydrocarbons, includingfor example, alkanes, such as decane, undecane, dodecane, tridecane,tetradecane, pentadecane, hexadecane; aliphatic and aromatic alkanessuch as dodecane, hexadecane, and cyclohexane, hydrocarbon oils, mineraloils, paraffin oils; halogenated oils, such as fluorocarbons andperfluorocarbons (e.g. 3M Fluorinert liquids) as well as mixtures of theabove. Examples of suitable gas filler fluids include, withoutlimitation, air, argon, nitrogen, carbon dioxide, oxygen, humidifiedair, any inert gases. In one embodiment, the primary phase is an aqueoussolution, and the secondary phase is air or oil, which is relativelyimmiscible with water. In another embodiment, the filler fluid includesa gas that fills the space between the plates surrounding the droplets.A preferred filler fluid is low-viscosity oil, such as silicone oil.Other suitable fluids are described in U.S. Patent Application No.60/736,399, entitled “Filler Fluids for Droplet-Based Microfluidics”filed on Nov. 14, 2005, the entire disclosure of which is incorporatedherein by reference. The fluid may be selected to prevent anysignificant evaporation of the droplets.

As will be understood by those in the art, the movement of droplets frompad to pad, with the addition of reagents as needed, can be used for anynumber of sample manipulations. In the case of the nucleic acidmanipulations for nucleic acid detection, these manipulations generallyinclude the addition of reagents, such as PCR enzymes, PCR buffer,primers, exonuclease, reverse transcriptase (RT) enzymes, RT-PCRbuffers, signal buffers, signal probes, etc.

In various embodiments, one or more portions, or sections, of theelectrode grid pathway of pads is/are exposed to heat within discretethermal zones for, e.g., amplification, exonuclease digestion, reversetranscription, target elution, and electrochemical detection. Suchthermal zones may comprise a detection region 378, an exonuclease region380, and a thermocycling (PCR) regions (also referred to as thermalzones) 382 a, 382 b, 382 c.

As will be appreciated by those in the art, some manipulations, such asPCR amplification, require the thermocycling between 2 to 3 differenttemperatures (primer binding, extension and denaturation), while othersrequire a uniform temperature for best results, e.g., enzymaticprocesses such as the use of exonuclease and reverse transcriptase,specific temperature(s) for improved elution and/or reagentresuspension, or binding/assay temperatures in the case of theelectrochemical detection. Isothermal amplification techniques and otherPCR alternatives typically require precise temperature control.

In various embodiments, heat applied to different portions of thefluidic processing panel 354 is generated by thermal components, such asresistive heaters or thermoelectric (Peltier) chips and are foundoff-cartridge in the processing bays of the instrument into which thecartridge 10 is placed. Examples of such thermal components aredescribed below.

In one embodiment, the sample manipulation zones on the reactor panel354 can optionally include sensors, for example, to monitor and controlthermal zone temperatures, particularly in the case where specifictemperatures are desirable. These sensors can include, but are notlimited to, thermocouples and resistance temperature detectors (RTDs).Alternatively, such sensors can also be “off cartridge” in the bays.

In various embodiments for detecting nucleic acid targets, the fluidicprocessing panel 354 comprises one or more thermocycling, or PCR oramplification, pathways 364 a, 364 b, 364 c, and 364 d. The fluidicprocessing panel 354 can contain 1, 2, 3 or more thermocycling pathwaysof pads. These can be used for individual PCR reactions (e.g., onedroplet is moved up and down a pathway or up one pathway and downanother, etc.) or for multiplexing (e.g. for multiple pathways, multipledifferent droplets can be moved up and down each pathway).

As will be appreciated by those in the art, each PCR reaction canadditionally be multiplexed. That is, for target-specific amplification,the use of multiple primer sets in a single PCR reaction can beunwieldy, and thus the present invention allows multiple reactions toachieve higher levels of multiplexing. For example, for the evaluationof 21 different target sequences (for example, in screening ofrespiratory viruses), it may be desirable to run 3 different reactionsof seven primer sets; e.g. a first PCR sample droplet in a first pathwaypicks up a first set of 7 primer pairs (e.g., “Primer Mix A”), a seconddroplet picks in a second pathway up a second set of 7 primer pairs(“Primer Mix B”), and a third droplet in a third pathway pick up a thirdset (“Primer Mix C”). In some embodiments, more than one droplet can beprocessed in each pathway, so each pathway may include more than oneprimer set. In some embodiments, the primers will be completelydifferent in each set; in others, redundancy and/or internal controlsare built into the system by adding the same primer sets to differentpathways. The number of multiplexes can vary easily through softwarewithout the need to modify any physical components of the system.

In general, amplification reactions suitable for use in the presentsystems use sets of primers wherein one primer of each set has a blockedend that is impervious to standard exonucleases. That is, it isdesirable to remove one strand of the double stranded amplicons that aregenerated in the PCR reaction, so as to simplify the detection reactionsand remove background signal. Thus, by running a first PCR reaction andthen adding exonuclease, one strand of the double stranded amplicon isdigested, leaving only the detection strand.

The use of multiple heating zones along the thermocycling pathways 364a-d, as generally depicted in FIG. 59, allows the droplets to travelthrough the appropriate thermal zones. As shown in FIG. 59, the fourthermocycling pathways 364 a, 364 b, 364 c, and 364 d are shown thatextend through the three thermal zones 382 a, 382 b, and 382 c. Thermalelements, e.g., resistive heaters, corresponding to the thermal zones,382 a, 382 b, and 382 c zones are off-cartridge heater elements and maybe maintained at temperatures of 95° C., 72° C., and 64° C. for use inPCR thermocycling. In some embodiments, two different temperature zones(e.g., about 95° C. for denaturation and about 60° C. for annealing andextension) can be used for a two-step PCR reaction. In otherembodiments, a three-zone, two-temperature configuration may beemployed, wherein a middle heater corresponding to middle thermal zone382 b controls the denaturation temperature (e.g., about 95° C.), andadditional heaters corresponding to the thermal zones 382 a, 382 c oneach side of the denaturation heater provide substantially the sameannealing and extension temperature (e.g., about 60° C.). In thisconfiguration, two-step amplification cycles can be performed with morethan one droplet in each thermocycling pathway 364 a-d. For example, twodroplets may be positioned in each thermocycling pathway and spaced insuch a way that when one droplet is in the denaturation zone 382 b, theother is in one of the combined annealing and extension zones 382 a or382 b, and vice versa. Each droplet may pick up amplification reagents(e.g., a primer cocktail) at locations, for example, at each end of athermocycling pathway, such as locations 366 a, 366 b of each of thethermocycling pathways 364 a-d. By shuttling the droplets in tandem backand forth between the denaturation and annealing/extension zones, onecan amplify both of them in the same amount of time it would normallytake to amplify a single droplet. In a four pathway configuration asshown, this means that eight droplet can be amplified simultaneouslyinstead of three.

In various embodiments, the multiplex cartridge 10 of the presentinvention relies on the use of electrodes and electrochemical labels forthe detection of target analytes. Generally, the surface of electrodeswithin each electrosensor array 363 a, 363 b, 363 c, and 363 d(optionally coated with a self-assembled monolayer (SAM)) has captureligands which bind the target. A second label ligand, which also bindsto the target, is included, such that in the presence of the target, thelabel ligand is bound near the surface of the electrode, and can bedetected electronically.

Thus, the detection zone of the fluidic processing panel 354 comprisesone or more separate arrays of detection electrodes 363 a, 363 b, 363 c,and 363 d within the respective electrosensor zones 360 a, 360 b, 360 cand 360 d. By “electrode” herein is meant a composition, which, whenconnected to an electronic device, is able to sense a current or chargeand convert it to a signal. Alternatively, an electrode can be definedas a composition which can apply a potential to and/or pass electrons toor from species in the solution. Preferred electrodes are known in theart and include, but are not limited to, certain metals and theiroxides, including gold; platinum; palladium; silicon; aluminum; metaloxide electrodes including platinum oxide, titanium oxide, tin oxide,indium tin oxide, palladium oxide, silicon oxide, aluminum oxide,molybdenum oxide (Mo₂0₆), tungsten oxide (W0₃) and ruthenium oxides; andcarbon (including glassy carbon electrodes, graphite and carbon paste).Preferred electrodes include gold, silicon, carbon and metal oxideelectrodes, with gold being particularly preferred. In a particularlyuseful embodiment, both the electrowetting electrode grid and thedetection electrodes are gold, and are fabricated simultaneously on thefluidic processing panel 354.

The present system finds particular utility in array formats, i.e.,wherein there is a matrix of addressable detection electrodes. By“array” herein is meant a plurality of capture ligands on electrodes inan array format; the size of the array will depend on the compositionand end use of the array. Arrays containing from about two differentcapture ligands to about 50 to 100 can be made. In some preferredembodiments, 80 or 100 working detection electrodes are split into fouror five distinct zones of twenty, with each zone having up to sixtycapture probes (three different capture probes per electrode).

The detection zone of the fluidic processing panel 354 comprises one ormore arrays of detection electrodes 363 a-d, each of which is within anelectrosensor zone 360 a-d that is in fluid communication with thedroplet pathway of an associated one of the detection mixing zones 385a-d. That is, the droplets containing the amplicons will pick upnecessary detection reagent such as label probe (e.g., a signal probecocktail which may be in dry form, e.g., at locations 362 a, 362 b, 362c, and 362 d) adjacent to the electrosensor detection zones 360 a, 360b, 360 c, and 360 d, respectively, and then be dispersed on theassociated electrosensor detection zones 360 a, 360 b, 360 c, and 360 d.The signal probe cocktails may be applied to a portion of the top plate241 forming the locations 362 a, 362 b, 362 c, and 362 d or a portion ofthe fluidic processing panel 354 covering the locations 362 a, 362 b,362 c, and 362 d. In general, each detection zone receives one or moresample droplets which are generally dispersed on the array ofelectrodes, which is considered one larger “pad”.

In one embodiment, the reaction module 240 includes four (4)electrosensor detection zones, and each electrosensor array includes 20working electrodes (which may include one reference electrode and oneauxiliary electrode). Each detection electrode of each electrosensorarray 363 a-d comprises an independent lead (interconnect) to transmitinput and electronic response signals for each electrode of the arraysuch that both input and electronic response signals are independentlymonitorable for each electrode. That is, each electrode is independentlyaddressable. Moreover, the reaction module is preferably configured forindependent control of electrowetting pads surrounding each electrode ofeach electrosensor array 363 a, 363 b, 363 c, and 363 d.

In addition to the components of the fluidic processing panel 354described above, the fluidic processing panel 354 can also optionallycomprise an EPROM, EEPROM or RFID to identify the cartridge, for examplecontaining information about the batch, treatment or contents of themultiplex cartridge 10. This can include information about theidentification of the assay, for example.

Instrument Overview

An instrument configured for processing the multiplex cartridge 10 andembodying aspects of the present invention is indicated by referencenumber 400 in FIG. 32. The instrument comprises a control console 402,one or more processing modules 410 operatively coupled to the controlconsole 402, processing bays within each processing module 410, each ofwhich is configured to receive a multiplex cartridge and process themultiplex cartridge independently of the other bays, and instrumentsoftware (ISW). In various embodiments, the instrument comprises onecontrol console 402 and up to four processing modules 410, with eachprocessing module including six processing bays. Each processing module410 is operatively coupled to the control console 402, e.g., to exchangepower, input and output data, and control signal transmissions with thecontrol console 402 and may be physically connected to the controlconsole 402 as well. Each processing bay with the processing module 410is configured to accept one multiplex cartridge 10 at a time and toprocess the cartridge independently of other processing bays processingother multiplex cartridges. In various embodiments, the instrument isconfigured for each processing bay to complete processing of a cartridgein 60 minutes or less.

The ISW provides the graphical user interface for the user to startruns, receive results, and provide inputs that at least partiallycontrol operation of the instrument. In various embodiments, the ISW isconfigured to run on a Windows® computer with a touchscreen 404 locatedon the control console 402 providing the primary functionality for userinput. In various embodiments, the instrument is configured to provideconnectivity to a local area network (“LAN”) and a laboratoryinformation system (“LIS”). The instrument may also include a barcodescanner (not shown) that facilitates logging in to the ISW, trackingsamples, and positive ID features of the instrument.

The control console 402 of the instrument includes a touchscreen panel404, a system computer, a power supply, connectivity to external datasystems, and connectivity for the processing module(s) and processingbay(s). In various embodiments, a power supply in the control consolepowers the entire instrument. Cabling from the control console providespower transmission and provides for data flow to and from the processingbays. In various embodiments, the control console also has provision forphysically attaching the one or more processing modules to the controlconsole

Each processing bay includes hardware, firmware, and electronics thatrun an assay on a multiplex cartridge 10. Each processing bay mayinclude a bay PCB. In various embodiments, the bay PCB includes theelectronics and firmware of the processing bay (such as, microprocessorsand firmware on the microprocessors), circuitry that supplies power(e.g., up to 300 V to the electrowetting pads) in the multiplexcartridge, circuitry that performs electronic sensing of reactionproducts on the multiplex cartridge, circuitry that controls heaters inthe processing bay that interact with the multiplex cartridge, circuitrythat measures and controls temperatures in the multiplex cartridge,circuitry that controls motion of various moving components of theprocessing bay, and circuitry that controls a pump of the processingbay.

Each processing bay may also include a connector PCB. In variousembodiments, the connector PCB includes pogo pins configured to makecontact with the multiplex cartridge and transmit data, control signals,and power between the multiplex cartridge and the processing bay PCB andpogo pins configured to make electrical contact with heater elementswithin the processing bay.

Each processing bay further includes stepper motors. In variousembodiments, the processing bay comprises two stepper motors: onestepper motor that controls positioning of magnets, heaters, and pogopins, or other connector elements, relative to the multiplex cartridge,and one stepper motor controls a cam follower plate within theprocessing bay that compresses blisters on the multiplex cartridge andcauses the blisters to dispense their contents in a predefined sequence.

Each processing bay also includes a blister compression assemblyconfigured to compress the blisters of the multiplex cartridge 10 in aspecified sequence and actuate the active valves of the multiplexcartridge 10, thereby dispensing the contents of the cartridge'sblisters in the specified sequence. In various embodiments, the blistercompression mechanism assembly comprises an array of blister-compressingactuators, or compression mechanisms, each comprising a cam armconfigured to push a compression pad onto a blister. The blistercompression mechanism assembly further includes a cam arm plate withinwhich the cam arms and compression pads of the compression mechanismsare operatively mounted above the blisters for movement between aretracted position and an extended, blister-compressing position, a camfollower plate that is movable with respect to the cam arm plate andincludes grooves with ridges (or other cam follow elements) located andsequenced to engage cam arms of the actuator array as the cam followerplate moves with respect to the cam arm plate to actuate the cam arms tocompress the blisters in a sequence determined by the relative locationsof the compression mechanisms in the cam arm plate and the grooves andridges of the cam follower plate.

Each processing bay may also include a pump coupled to the multiplexcartridge 10 via pump port 104 and configured to provide a motivatingforce for reagents and sample in sample preparation module of themultiplex cartridge.

Each processing bay may also include an LED PCB 466 (see FIGS. 38-41)that provides LED indicators of the processing bay status and opticalsensors that detect conditions within the multiplex cartridge, forexample, through inlet optical port 14 and outlet optical port 16.

Each processing bay may also include mounting hardware configured toattach the processing bay into the processing module and electricalconnectors configured to transmit power and data between the processingbay and the processing module.

Each processing bay may also include a multiplex cartridge carrierconfigured to provide a physical connection and alignment between thetop bay, comprising the blister compression mechanism assembly, and amultiplex cartridge processing assembly, or bottom bay, comprising acartridge carriage assembly, a heating and control assembly, and a camframe assembly configured to effect movement of the heating and controlassembly with respect to a multiplex cartridge held in the cartridgecarriage assembly.

Control Console

A processing instrument embodying aspects of the present invention andconfigured to process the multiplex cartridge 10 described above isindicated by reference number 400 in FIG. 32. As noted above, theinstrument 400 includes the control console 402 and one or moreprocessing modules 410 operatively associated with the control console402. The control console 402, in one embodiment, includes a displaypanel 404 presenting a graphical user interface and comprising atouchscreen by which a user may input information to the control console402 and/or by which information can be presented to the user. In variousembodiments, the control console 402 may comprise additional oralternate means for inputting data, such as keyboards, microphones,switches, manually-operated scanners, voice-activated input, etc. Asfurther noted above, the instrument may include a barcode scanner forreading barcodes, for example, one-dimensional or two-dimensionalbarcodes, or other types of scanners for reading machine-readable code,such as an RFID scanner. In various embodiments, the control console 402may comprise additional or alternate means for outputting data (i.e.,information and/or results), including hard drives or other storagemedia, monitors, printers, indicator lights, or audible signal elements(e.g., buzzer, horn, bell, etc.), email, text message, etc.

Processing Module

As shown in FIGS. 32 and 34, each processing module 410 includes one ormore cartridge doors 412, each cartridge door 412 being associated witha processing bay (described below) within which a cartridge 10 may beprocessed. In the illustrated embodiment, each processing module 410includes six (6) cartridge doors 412 and associated processing bays.Each cartridge door 412 is configured to accept a multiplex cartridge10, preferably in a single, preferred orientation. Each cartridge dooralso preferably includes a closeable door (e.g., a pivoting door panel)that is biased, e.g., by a spring or the like, in a closed position butcan be pushed open when a cartridge is inserted therein.

In various embodiments, each processing module 410 is operativelycoupled to the control console 402. The processing module 410 may beelectronically coupled to the control console 402 so as to enableelectronic transmissions between the control console 402 and theprocessing module 410. Such electronic transmissions may comprise powertransmissions from the control console to the processing module forpowering various electronic components within the processing module,control signals, input data, output data, etc.

Each processing module 410 may also be physically connected, e.g., in aside-by-side relationship as shown in FIG. 32, with the control console402. As in the illustrated embodiment, the instrument 400 may includeone or more processing modules 410 secured to one or both sides of thecontrol console 402. Additional processing modules may be secured toother processing modules in a side-by-side relationship on one or bothsides of the control console 402. In one preferred arrangement, theinstrument 400 includes up to 2 processing modules 410 secured to eachside of the control console 402, each processing module 410 comprisingsix (6) cartridge doors 412 and associated processing bays forprocessing up to six multiplex cartridges 10 per processing module.

It is preferred that the control console 402 and the processing module410 be provided in a modular manner as shown so as to facilitatescalability of the instrument, e.g., by adding one or more processingmodules 410 to or subtracting one or more processing modules 410 from asingle control console 402, and also to facilitate instrumenttrouble-shooting whereby a processing module 410 having one or moremalfunctioning processing bays can be removed from the instrument forrepair or replacement, and the instrument may still be useable with theremaining, operative processing modules 410.

In an alternate embodiment, however, a control console and associatedinput screen—and/or other input means—and one or more—preferably aplurality of—cartridge doors and associate processing bays may beprovided in a single, integral instrument having a single housing.

Further details of the processing module 410 are shown in FIGS. 35, 36,and 37. Each processing module 410 includes a plurality of cartridgedoors 412 and associated processing bays 440. The illustrated embodimentincludes six (6) processing bays 440. The processing bays 440 arearranged in a stacked arrangement within a housing of the processingmodule 410. Each processing bay 440 has associated therewith a frame 418partially surrounding the processing bay with a horizontal top panel 420and a vertical rear panel 422 (See FIG. 37). As shown in the figures, afront panel 413 of the processing module 410 within which the cartridgedoors 412 are positioned is oriented at an angle tilted back from thebottom of the processing module 410 to the top of the processing module410. This may be for ergonomic and/or esthetic reasons. In otherembodiments, a front panel of the processing module may be vertical.Because of the angle of the front panel 413 of the processing module410, each processing bay 440 is offset horizontally (i.e., rearwardly)relative to the processing bay immediately below it.

In various embodiments, each processing bay 440 has associated therewitha ventilation fan 416 secured to the vertical panel 422 of the housing418 and a ventilation duct 414 extending from the fan 416 to a rear wallof the housing of the processing module 410. As shown in the figures,due to the tilt of the front panel 413 and the horizontal offset of theprocessing bays 440, the ventilation ducts 414 have decreasing lengthsprogressing from the bottom-most processing bay 440 to the top-mostprocessing bay.

The processing module 410 may further include additional structuralelements for securing each of the processing bays 440 within the housingof the processing module. The processing bays 440 and processing module410 are preferably configured so that each bay 440 may be independentlyremoved from the processing module 410 and replaced to facilitateinstrument repair if one or more processing bays 440 malfunctions or isotherwise in need of maintenance or repair.

Processing Bay

A processing bay 440 is shown in various views in FIGS. 38, 39, 40, and41. In each of FIGS. 38-41, the frame 418 of the processing bay 440 isomitted from the figure. FIG. 38 is a front, right-side perspective viewof the processing module 440 with a multiplex cartridge 10 insertedtherein. FIG. 39 is a front, left-side perspective view of theprocessing module 440 with a multiplex cartridge 10 inserted therein.FIG. 40 is a rear, right-side perspective view of the processing module440. FIG. 41 is a front, right-side, exploded perspective view of theprocessing module 440 with a multiplex cartridge 10 inserted therein.

Each processing bay 440 has a drip tray 446 forming a lower floor of theprocessing bay 440 and constructed and arranged to contain fluid leaksthat may occur from the multiplex cartridge 10 and to provide a supportand mounting structure for various components of the processing bay 440.A main PCB (printed circuit board) 442, also referred to as the bay PCB,provides primary control of the processing bay 440 as well as data andpower distribution and transmission. A flexible connector 444 connectsthe bay PCB 442 with a connector PCB (described below, not visible inFIGS. 38-41) within the processing bay 440, as will be described infurther detail below. The processing bay 440 may further includealignment elements, such as two (2) tubular female alignment elements460, 462, that receive male alignment elements disposed within theprocessing module 410 for properly aligning and positioning theprocessing bay 440 in a bay mounting location within the processingmodule 410.

The processing bay 440 may be conceptually divided along functionallines between a cartridge processing assembly 470 (also known as thelower bay) and a blister (or deformable chamber) compression mechanismassembly 750 (also known as the upper bay). The primary function of thecartridge processing assembly 470 is to receive the cartridge 10, securethe cartridge within the bay 440, apply heat and magnetic forces to theprocessing module 240 of the multiplex cartridge 10, apply controlledpower to the multiplex cartridge 10, engage the rotary mixer 192 of thecartridge 10 and effect powered rotation of the rotary mixer 192, andeject the cartridge 10 from the processing bay 440 at the conclusion ofan assay or other process performed within the bay 440. The primaryfunction of the blister compression mechanism assembly 750 is tocollapse the various deformable chambers of the multiplex cartridge 10in a proper sequence. Each of these various components will be discussedin further detail below.

Processing bay 440 further includes an LED PCB 466 for controlling oneor more LEDs that provide information to a user, such as indicating thestatus of the processing bay 440 and/or whether a cartridge is locatedwithin the processing bay 440. The status LEDs may be visible via alight pipe or other optical transmitter that provides an opticalindication signal adjacent to the cartridge door 412 associated with thebay 440 on the front panel 413 of the processing module 410. The LED PCB466 may also control optical sensors constructed and arranged to detect(e.g., generate a signal), through the inlet and outlet optical ports14, 16, fluid flow through the inlet optical sensing chamber 154 and theoutlet optical sensing chamber 158 of the sample preparation module 70.

Sidewalls 472, 474 extend upwardly along opposite sides of theprocessing bay 440 and may be secured to upwardly extending elements 443445 of the drip tray 446. A mounting plate 640 includes a generallyhorizontal blister plate 644 (see FIG. 42) secured to the top edges ofthe sidewalls 472, 474 and which generally separates the cartridgeprocessing assembly 470 from the blister compression assembly 750.

In various embodiments, each processing bay 440 further includes a camfollower motor 834 and an associated encoder 838 and a cam frame motor602. The cam plate motor 834 and the cam frame motor 602 are secured toa motor mount 642 of the mounting plate 640 (see FIG. 42).

A pump 458 provides the pressure that is applied to the multiplexcartridge 10 via the pump port 104.

As will be described in further detail below, the cartridge processingassembly 470 includes a Peltier heater assembly for effecting thermalprocesses within the processing bay 440. To ventilate the processing bay440 and dissipate excess heat generated at the Peltier heater, theprocessing bay 440 may include a peltier ventilation assembly. Theventilation assembly comprises a cooling fan 448 attached to a fan mount450 of the drip tray 446 and positioned in front of an airflow ductextending between the cooling fan 448 and the Peltier heating assemblywithin the processing bay 440. In various embodiments, the airflow ductmay comprise a cooling duct 452 and a duct cover 456 extending betweenthe cooling fan 448 and the beginning of the cooling duct 452. (SeeFIGS. 39 and 41).

Cartridge Processing Assembly (Lower Bay)

Aspects of the cartridge processing assembly 470 are shown in FIGS. 41and 42. As noted above, most features of the cartridge processingassembly 470 are located beneath the blister plate 644 of the motormount 642. The cartridge processing assembly 470 includes a cartridgecarriage assembly 650 configured to receive and hold, and later eject, amultiplex cartridge 10. The cartridge carriage assembly 650 is securedto a bottom surface of the blister plate 644 of the mounting plate 640.

A cam block assembly 600 includes a cam frame 606 that surrounds thecartridge carriage assembly 650 on three sides and is mounted for linearfore and aft movement within the processing bay 440 where it issupported on linear cam followers 480 a, 480 b extending from each ofthe sidewalls 472, 474 into a follower slot 612 formed on each side ofthe cam frame 606.

A mixing motor assembly 700 is pivotally connected to the blister plate644 beneath the blister plate and is configured to pivot into and out ofan operative engagement with the rotary mixer 192 of the multiplexcartridge 10 disposed within the cartridge carriage assembly 650.

A heating and control assembly 500 is positioned beneath the cartridgecarriage assembly 650 and is operatively coupled to the cam frame 606and the cam block assembly 600 for converting the longitudinal, fore andaft movement of the cam frame 606 into vertical movement of the heatingand control assembly 500 for selectively bringing the heating andcontrol assembly 500 into contact with a bottom surface of the multiplexcartridge 10 when a cartridge is inserted into the cartridge carriageassembly 650.

Cartridge Carriage Assembly

Further details of the cartridge carriage assembly 650 are shown in FIG.46, which is an exploded perspective view of the cartridge carriageassembly 650 with other components of the cartridge processing assembly470 omitted. The cartridge carriage assembly 650 includes a carriageholder 652 comprising a generally rectangular frame which is secured tothe underside of the blister plate 644 of the mounting plate 640. Adetector may be provided for detecting when a multiplex cartridge 10(not shown in FIG. 46) is inserted into the cartridge holder 652. Invarious embodiments, the detector comprises an optical detectorcomprising an emitter 686 and detector 688 each disposed within arespective pocket on opposite sides of the cartridge holder 652. Anoptical beam from the emitter 686 to the detector 688 is broken when amultiplex cartridge is inserted into the cartridge holder 652, therebygenerating a signal indicating the presence of the cartridge.

A cartridge latch 654 is mounted for pivotal movement at a closed end ofthe cartridge holder 652. The cartridge latch 654 is pivotally mountedon a latch pin 660 for rotation about a horizontal axis of rotation. Thecartridge latch 654 further includes a forward hook 656 and a trailinglever 658. A torsion spring 662 rotationally biases the latch 654 sothat the hook 656 is in an upward position. When a cartridge 10 isinserted into the cartridge holder 652, the cartridge pushes the hookdown until the hook 656 of the cartridge latch 654 engages a recess in abottom portion of the lower shroud 30 of the cartridge 10. The bias ofthe torsion spring 662 holds the hook 656 into that recess to retain thecartridge within the cartridge holder 652.

A cartridge ejector assembly 670 includes an ejector rack 672 that ispositioned within an ejector bracket 682 extending off a rear end of thecartridge holder 652. The linear gear-teeth of the ejector rack 672engage a damper pinion gear 674 that is coupled to a rotary damper 676and is mounted for rotation on the ejector bracket 682 adjacent theejector rack 672. A spring capture pin 680 extends through the ejectorrack 672 and is supported at an end thereof by an end wall of theejector bracket 682. A compression spring 678 is disposed between an endof the ejector rack 672 and the end of the spring capture pin 680.Accordingly, the ejector rack 672 is biased longitudinally toward theopen end of the cartridge holder 652. A limit stop element may beprovided to prevent the cartridge rack 672 from being pushed too far bythe spring 678. The ejector rack 672 initially extends into thecartridge holder 652 and is contacted by the end of a multiplexcartridge 10 inserted into the cartridge holder 652. As the cartridge isfurther inserted into the cartridge holder 652, the ejector rack 672 ispushed back, thereby compressing the spring 678 and generating a biasforce urging the cartridge 10 longitudinally toward the open end of thecartridge holder 652 and out of the processing bay 440. Because thecartridge latch 654 captures the fully-inserted multiplex cartridge, theejector assembly 670 is prevented from pushing the cartridge back out ofthe cartridge holder 652.

A cartridge latch switch 666 is positioned at the closed end of thecartridge holder 652 and is configured to signal when the multiplexcartridge has been inserted to a position within the cartridge holder652, such that the cartridge will be engaged by the cartridge latch 654.At the conclusion of an assay or other process performed within theprocessing bay 440 the cartridge latch 654 is pivoted (counterclockwisein the illustrated embodiment) against the bias of the torsion spring662, in a manner that will be described below, to thereby release themultiplex cartridge held within the cartridge holder 652. Upon releaseof the cartridge, the cartridge is ejected by the stored energy in thecompress spring 678 bearing against the ejector rack 672. The damperpinion 674 and the operatively-associated rotary damper 676 with whichthe ejector rack 672 is engaged ensures a controlled release of theejector rack 672 so that the multiplex cartridge 10 is not ejected tooabruptly from the cartridge holder 652.

Heating and Control Assembly

Details of the heating and control assembly 500 are shown in FIG. 43,which is an exploded perspective view of the heating and controlassembly 500 with other components of the cartridge processing assembly470 omitted.

The heating and control assembly 500 includes a support plate 502, aconnector PCB 504 supported on the support plate 502, a cover plate 550partially covering the connector PCB 504, a cartridge magnet assembly552, a sample preparation magnet assembly 570, and a magnet actuator 584located beneath the support plate 502. A front alignment pin 416 and arear alignment pin 414 extend upwardly from the support plate 502.

A pneumatic connector 518 is attached to pneumatic ports 519 a, 519 b ofthe cover plate 550. The pneumatic connector 518 provides a connectionbetween the pressure source, e.g., pump 458, and the cartridge 10 viapump port 104 and provides a connection between an external valve withinthe processing bay 440 and the passive valve assembly 220 of thecartridge 10 via the passive valve port 108 (see FIG. 15).

Referring to FIGS. 43 and 44, which is a top plan view of the connectorPCB 504, the connector PCB 504 includes an elution heater assembly 506,a detection Peltier assembly 540, and PCR heater assembly 520 a, 520 b,and 520 c. In various embodiments, the elution heater assembly 506comprises a resistive heating element attached to a dedicated PCB and aheat spreader comprised of a thermally-conductive material attached orotherwise thermally coupled to the resistive heating element. Similarly,in various embodiments, each element 520 a, 520 b, and 520 c of the PCRheater assembly comprises a resistive heating element attached to adedicated PCB and a heat spreader comprised of a thermally-conductivematerial attached or otherwise thermally coupled to the resistiveheating element.

Details of the detection Peltier assembly 540 are show in FIG. 45, whichis an exploded, perspective view of the Peltier assembly 540. Theassembly 540 includes a Peltier device 544 (i.e., a thermoelectricelement) coupled to a power and control printed circuit board 546. Aheat spreader 542, preferably comprised of a thermally conductivematerial, is disposed above the Peltier device 544. A heat sink 548 isdisposed beneath the peltier chip 544. The heat sink 548 may comprise apanel that is in surface-to-surface contact with a surface of thePeltier device 544 with a plurality of heat-dissipating rods (or fins)extending therefrom and formed from a thermally conductive material. Thedetection Peltier assembly 540 is mounted within, and at least a portionof the heat sink 548 extends through, an associated opening formed inthe support plate 542. The heat dissipating rods of the heat sink 548extend beneath the support plate 502 and are disposed at a terminal endof the Peltier cooling duct 452 (See FIGS. 39 and 41). In oneembodiment, the detection Peltier is configured to apply a thermalgradient to, e.g., reduce the temperature of, a detection area, e.g.,the detection region 378, of the multiplex cartridge 10.

A plurality of connector pin arrays 510 a, 510 b, 510 c, 510 d, 510 d,510 e, 510 f, and 510 g are disposed around the connector PCB 504 andcomprise arrays of connector pogo pins that contact and effectelectrical connection between connection pads of associated connectorpad arrays 358 a-358 g of the fluidic processing panel 354 of themultiplex cartridge 10 (See FIG. 58). Connections between the connectorpin arrays 510 a-510 g and the connector pad arrays 358 a-358 g providesconnections between the instrument 400 and the multiplex cartridge 10for, e.g., power, control signals, and data. For example, theconnections between the connector pin arrays 510 a-510 g and theconnector pad arrays 358 a-358 g provides provide power and control fromthe instrument to the electrowetting grid (e.g., the thermal cyclingtracks 364 a-364 d, the sample bead zone 368, the hybridization zone370, the elution buffer zone 372, the exonuclease reagent zone 374, thePCR reagent zone 376, the detection mixing zones 385 a-385 d, and theexonuclease zone 384). In addition, connections between the connectorpin arrays 510 a-510 g and the connector pad arrays 358 a-358 g providespower to and receives date from the electrosensor arrays 363 a-363 d.

As shown in FIG. 43, the connector PCB 504 further includes a number ofheater pins 512—which may comprise pogo pins—that connect to the variousheater assemblies 540, 506, and 520 a, b, c.

The heating and control assembly 500 further includes a cartridge magnetassembly 552 and a sample preparation magnet assembly 570.

Details of the sample preparation magnet assembly 570 are shown in FIG.49A, which is a top perspective view of the sample preparation magnetassembly. The sample preparation magnet assembly 570 comprises a magnetholder 572 mounted on a horizontal spindle 574 so as to be rotatableabout the spindle 574 relative to the support plate 502. A torsionspring 576 biases the sample preparation magnet assembly 570 downwardly.An actuator bracket 578 extends beneath the magnet holder 572, and amagnet 580 is supported on top of the magnet holder 572 and is securedthereto, e.g., by a suitable adhesive. When deployed and rotatedupwardly against the bias of the torsion spring 576, the magnet 580extends through aligned openings formed in the support plate 502, theconnector PCB 504, and the cover plate 550.

The sample preparation magnet assembly 570, when deployed, is positionedadjacent the capture chamber 100 of the sample preparation module 70 ofthe multiplex cartridge 10 to thereby apply a magnetic force to fluidscontained within and flowing through the capture chamber.

Details of the cartridge magnet assembly 552 are shown in FIG. 49B,which is a top perspective view of the cartridge magnet assembly. Thecartridge magnet assembly 552 comprises a magnet holder frame 554 and amagnet array 556 disposed within the magnet holder frame 554. The magnetarray 556 may comprise individual magnets (e.g., three), and may besurrounded on four sides by the magnet holder frame 554 to form a framesurrounding the magnet array 556. The magnet array 556 may be securedwithin the magnet holder frame 554 by, for example, a suitable adhesive.A focusing magnet 558 is disposed within an opening in a top part of theframe of the magnet holder 554. In one embodiment, the focusing magnet558 is cylindrical and may comprise neodymium N52. The focusing magnet558 focuses the magnetic forces of the magnet array 556 to a relativelysmall area for attracting magnetic target capture beads to that smallarea. The magnet holder 554 is mounted on a horizontal spindle 560connected to the support plate 502 so that the magnet holder 554 and themagnet array 556 are rotatable about the spindle 560. A torsion spring562 biases the cartridge magnet assembly 552 downwardly. An actuatorbracket 566 extends beneath the magnet holder 554. When the magnetholder 554 is rotated upwardly against the bias of the torsion spring562, the upper portion of the magnet assembly 552 extends throughaligned openings formed in the support plate 502, the connector PCB 504,and the cover plate 550.

The cartridge magnet assembly 552, when deployed, is positioned adjacentto the sample chamber 266 of the reaction module 240, adjacent to aposition indicated by reference number 270 (see FIG. 26).

Returning now to FIG. 43, cam followers 590 a and 590 b extend fromopposite sides of the support plate 502 and a slot follower 592 extendsfrom opposite sides of the support plate 502. The slot followers 592extend into and are vertically movable within a slot 476 formed in eachof the side walls 472, 474 (see FIG. 42) and are configured to enablevertical movement of the support plate 502 relative to the side walls472, 474 while preventing horizontal movement of the support plate 502relative to the side walls 472, 474.

Cam Frame Assembly

Details of a cam frame assembly 600 are shown in FIG. 47, which is anexploded perspective view of the cam frame assembly 600 with othercomponents of the cartridge processing assembly 470 omitted. The camframe assembly 600 includes the cam frame motor 602 that drives a linearactuator 604. A cam frame 606 includes opposed, generally parallel,longitudinal spars 608, 610 and a cross spar 614 extending betweencorresponding ends of each of the longitudinal spars 608, 610. Thelinear actuator 604 is coupled to the cam frame 606 at a motor connector618 projecting upwardly from the cross spar 614. A follower slot, orchannel, 612 is formed along the outer side beneath a top surface of theeach of the longitudinal spars 608, 610. Follower elements 480 a, 480 bextending from each of the side walls 472, 474 (See FIG. 42) extend intothe follower slot 612.

A cam rail 620 a is secured to the longitudinal spar 608, and a cam rail620 b is secured to the longitudinal spar 610. A top edge of the camrail 620 a cooperates with the follower slot 612 formed in lower outeredge of the longitudinal spar 608 to form a channel that receives thecam followers 480 a, 480 b, which permit longitudinal movement of thecam frame 606 and the cam rails 620 a, 620 b with respect to the sidewalls 472, 474, while preventing vertical movement of the cam frame 606relative to the side walls 472, 474.

Each cam rail 620 a and 620 b includes a forward cam slot 622 a and arear cam slot 622 b. The cam followers 590 a, 590 b projecting from theside of the support plate 502 of the heating and control assembly 500(See FIG. 43) extend into the cam slots 622 a, 622 b, respectively. Eachcam slot 622 a, 622 b has a lower horizontal segment (the right-sidesegment in FIG. 47), an upper horizontal segment (the left-side segmentin FIG. 47), and an angled transition between the lower horizontalsegment and the upper horizontal segment.

Before a multiplex cartridge 10 is inserted into the cartridge carriageassembly 650, the cam frame 606 is in a relatively forward positionrelative to the heating and control assembly 500 so that the camfollowers 590 a, 590 b extending from the support plate 502 are at thelower horizontal segment (the right side segment as shown in FIG. 47) ofeach of the cam slots 622 a, 622 b. Thus, the support plate 502 and theentire heating and control assembly 500 is in a down position withrespect to the cartridge carriage assembly 650. When a multiplexcartridge 10 is inserted into the cartridge carriage assembly 650, thealignment fork 246 of the top plate 241 (see FIG. 24) engages the rearalignment pin 514—which is longer than the front alignment pin 516 andextends up into the cartridge carriage assembly 650 even with thesupport plate 502 in the down position—to properly position thecartridge within the carriage assembly 650.

After the multiplex cartridge is inserted into the cartridge carriageassembly 650, as indicated, for example, when the cartridge latch switch666 is triggered by the end of a fully-inserted cartridge, the cam framemotor 602 is activated to retract the linear actuator 604 and the camframe 606 attached thereto. This causes movement of cam rails 620 a, 620b relative to the support plate 502, thereby moving the cam followers590 a, 590 b from the lower, right side horizontal segments of the camslots 622 a, 622 b, up the angled transitions, and to the upper, leftside horizontal segments of the cam slots 622 a, 622 b, thereby raisingthe support plate 502 and the heating and control assembly 500 intocontact with the multiplex cartridge that has been placed into thecartridge carriage assembly 650.

Raising the support plate 502 relative to the cartridge held in thecartridge carriage assembly 650, causes the front alignment pin 516 ofthe support plate 502 to extend into the alignment loop 244 extendingfrom the top plate 241 (See FIG. 24). With the rear alignment pin 514engaged by the alignment fork 246 and the front alignment pin 516extending into the alignment loop 244, the cartridge is substantiallyimmobilized within the cartridge carriage assembly 650.

Raising the heating and control assembly 500 with respect to thecartridge 10 held in the cartridge carriage assembly 650 places theconnector pin arrays 510 a-510 g of the connector PCB 504 into contactwith the respective connector pad arrays 358 a-358 g of the fluidicprocessing panel 354 of the multiplex cartridge 10. In addition, theelution heater assembly 506 of the connector PCB 504 is brought intocontact or close proximity (i.e., so as to enable the transfer ofthermal energy) with a portion of the fluidic processing panel 354corresponding to the exonuclease region 380. Similarly, the componentsof the PCR heater assembly 520 a, 520 b, 520 c of the connector PCB 504are brought into contact or close proximity (i.e., so as to enable thetransfer of thermal energy) with portions of the fluidic processingpanel 354 corresponding to the thermocycling regions 382 a, 382 b, and382 c. The detection Peltier assembly 540 of the connector PCB 504 isbrought into contact or close proximity (i.e., so as to enable thetransfer of thermal energy) with portions of the fluidic processingpanel 354 corresponding to the detection region 378. Also, the pneumaticconnector 518 is brought into contact with the pump port 104 and thepassive valve port 108 of the sample preparation module 70 of themultiplex cartridge 10.

Each cam rail 620 a, 620 b is secured to the respective longitudinalspar 608, 610 of the cam frame 606 by means of two threaded springcapture posts 624 a, 624 b with a compression spring 626 a, 626 bdisposed between the cam rail 620 a and a head of each of the posts 624a, 624 b. This “shock absorber” configuration permits a certain amountof movement of the cam rails 620 a, 620 b relative to the longitudinalspars 608, 610 to thereby prevent the heating and control assembly 500from being pushed against the bottom of the multiplex cartridge 10 withtoo great a force. Accordingly, the heating and control assembly 500will be pushed against the bottom of the multiplex cartridge with aforce that is no greater than the compressive force of the springs 626a, 626 b.

Referring to FIGS. 43 and 48, which is a perspective cross-sectionalview of the cam frame and a magnet actuator 584 of the cartridgeprocessing assembly 470, a magnet actuator 584 is coupled to the camframe 606 and is configured to rotate the cartridge magnet assembly 552and the sample preparation magnet assembly 570 into their respectiveoperative positions with respect to a multiplex cartridge when thecartridge is inserted in the cartridge carriage assembly. The magnetactuator 584 includes a spring 587 that biases the actuator to the leftin FIG. 48. The magnet actuator 584 includes a vertical tab 585configured to engage the actuator bracket 566 of the cartridge magnetassembly 552 and a vertical tab 586 configured to engage the actuatorbracket 578 of the sample preparation magnet assembly 570. The magnetactuator 584 is coupled to the cam frame 606 by means of a magnetactuator hook 628 extending below the cross bar 614 and engaging a hookloop 589 formed in an end of the magnet actuator 584.

As noted above, before a multiplex cartridge 10 is inserted into thecartridge carriage assembly 650, the cam frame 606 is in a forwardposition. The magnet actuator 584 is biased forward (to the left) by thespring 587 so that the cartridge magnet assembly 552 and the samplepreparation magnet assembly 570 are rotated clockwise to their retractedpositions due to the force of their respective torsion springs 562, 576,respectively. In the present context, the retracted positions of thecartridge magnet assembly 552 and the sample preparation magnet assembly570 positions in which the cartridge magnet assembly 552 and the samplepreparation magnet assembly 570 do not apply a significant magneticforce to any portion of the multiplex cartridge 10. After the multiplexcartridge is inserted into the cartridge carriage assembly 650, the camframe 606 is retracted by the cam frame motor 602 and the linearactuator 604 (to the right in FIG. 48) as described above. Retraction ofthe cam frame 606 causes the heating and control assembly 500 to beraised into contact with the multiplex cartridge 10, as the camfollowers 590 a, 590 b of the support plate 502 move from the lower,right side horizontal segments of the cam slots 622 a, 622 b, up theangled transitions, and to the upper, left side horizontal segments ofthe cam slots 622 a, 622 b.

The magnet actuator 584 coupled to the cam frame 606 by the magnetactuator hook 628 also moves with the cam frame 606 to pull the magnetactuator 584 to the right in FIG. 48 against the bias of the spring 587.As the actuator bracket 584 is pulled by the moving cam frame 606, thevertical tab 585 engaging the actuator bracket 566 of the cartridgemagnet assembly 552 rotates the magnet assembly 552 counterclockwisetoward its upward, deployed position as shown in FIG. 48. Similarly, thevertical tab 586 of the actuator bracket 584 engaging the actuatorbracket 578 of the cartridge magnet assembly 570 rotates the magnetassembly 570 counterclockwise toward its upward, deployed position asshown in FIG. 48. Due to the longitudinal extent of the upper horizontalsegment of each of the cam slots 622 a, 622 b, the cam frame 606 and thecam rails 620 a, 620 b can move longitudinally with respect to thesupport plate 502, while the cam followers 590 a, 590 b are positionedin the upper horizontal segments, without changing the height positionof the support plate 502 and the heating and control assembly 500 withrespect to the multiplex cartridge that has been placed into thecartridge carriage assembly 650. In various embodiments, the magnetactuator bracket 584 is configured with respect to the cartridge magnetassembly 552 and the sample preparation magnet assembly 570 so that asthe cam frame 606 moves (to the right) to raise the support plate 502and the heating and control assembly 500, the magnet assemblies 552, 570are not initially deployed (or are not fully deployed) when the supportplate 502 and the heating and control assembly 500 are first raised intocontact with the multiplex cartridge (i.e., when the cam followers 590a, 590 b of the support plate 502 first reach the upper horizontalsegments of the cam slots 622 a, 622 b). Further movement (to the right)of the cam frame 606 (which, due to the longitudinal extent of the upperhorizontal segments of the cam slots 622 a, 622 b, will not change theposition of the support plate 502 and the heating and control assembly500 with respect to the cartridge carriage assembly 650 and themultiplex cartridge held therein) will further pull the magnet actuatorbracket 584 to fully rotate the magnet assemblies 552, 570(counter-clockwise) into their fully deployed positions in contact orclose proximity to the multiplex cartridge. Thus, with the support plate502 and the heating and control assembly 500 maintained at the upposition in contact with the multiplex cartridge, the magnet assembliesare configured for movement independently of the rest of the heating andcontrol assembly 500 and the cam frame 606 can move longitudinally toeffect selective deployment of the magnet assemblies 552, 570 in supportof requirements to selectively apply or remove magnetic forces withrespect to the multiplex cartridge held within the cartridge carriageassembly 650.

Also, as can be best seen in FIG. 48, when the cam frame 606 is advanced(to the left in FIG. 48) to lower the heating and control assembly 500relative to the cartridge, the linear actuator connector 618 extendingabove the cross bar 614 contacts the lever 658 of the cartridge latch654, thereby rotating the cartridge latch 654 counterclockwise to lowerthe hook 656 and disengage the hook 656 from the multiplex cartridge sothat the multiplex cartridge can be ejected from the cartridge holder652 by the cartridge ejector assembly 670.

Mixing Motor Assembly

Details of the mixing motor assembly 700 are shown in FIGS. 50A and 50B.FIG. 50A is a perspective view of the mixing motor assembly 700, andFIG. 50B is an exploded perspective view of the mixing motor assembly700.

The mixing motor assembly 700 includes a mixing motor bracket 702 towhich is mounted a mixing motor 706. Suitable motors include the PololuMicro Metal Gearmotor with a 150:1 gearbox and the Maxon, model DCX10LEB SL 4.5V with a 64:1 gearbox. Preferred characteristics of the motorinclude 100 rep at 12 oz-in torque, 3000 hrs. life at 45° C. operatingenvironment and compact size (e.g., 10 mm width (diameter) and less than25 mm long).

A beveled gear 708 is fixed to an output shaft of the motor 706. Abevel-spur gear 710 rotatably mounted to the mixing motor mountingbracket 702 is operatively coupled to the beveled gear 708 with thebeveled gear teeth of the bevel-spur gear 706 engaged with the beveledgear teeth of the beveled gear 708. Thus, powered rotation of thebeveled gear 708 about a horizontal axis of rotation corresponding tothe output shaft of the motor 706 is converted to a rotation of thebevel-spur gear 710 about a vertical axis of rotation.

The mixing motor assembly 700 is pivotally connected to an underside ofthe blister plate 644 of the mounting plate 640 by means of a pivotscrew 716 extending through the mixing motor bracket 702. A standoff 714(comprising a threaded screw and a cylindrical sleeve disposed over aportion of the screw shaft) is attached to one end of the mountingbracket 702. A torsion spring 718 is coupled to the pivot screw 716 andbiases the mixing motor assembly 700 inwardly relative to the sidewall474 (see FIG. 42) so that the bevel-spur gear 710 engages the peripheralgear teeth 198 of the rotary mixer 192 (see FIG. 8) of the multiplexcartridge 10.

As shown in FIG. 48, the longitudinal spar 610 of the cam frame 606includes a beveled block 616 extending inwardly from the longitudinalspar 610. As noted above, the mixing motor assembly 700 is biased topivot inwardly relative to the side wall 474 and the longitudinal spar610 due to the torsion spring 718. The beveled block 616 is positionedso as to engage the mixing motor assembly 700 when the cam frame 606 isin the forward position. Thus, when the cam frame 606 is in theretracted position to raise the heating and control assembly 500 intoengagement with the multiplex cartridge 10 held in the cartridgecarriage assembly 650, the mixing motor assembly 700 pivots inwardlyunder the force of the torsion spring 718 into engagement with themultiplex cartridge. As the cam frame 606 moves forwardly (to the leftin FIG. 48) to lower the heating and control assembly 500 away from themultiplex cartridge held in the cartridge carriage assembly 650, thebeveled block 616 contacts the standoff 714 of the mixing motor assembly700 and pivots the mixing motor assembly outwardly (toward thelongitudinal spar 610) against the bias of the torsion spring 718 todisengage the bevel spur gear 710 from the rotary mixer 192 of themultiplex cartridge 10. In one embodiment, the beveled block 616contacts the standoff 714 to pivot the mixing motor assembly 700 out ofengagement with the rotary mixer 192 before the actuator connector 618of the cam frame 606 contacts the lever 658 of the cartridge latch 654to lower the hook 656 and release the cartridge to be ejected by thecartridge ejector assembly 670.

Thus, when the cam frame 606 is in the forward position, the heating andcontrol panel 500 is in the lowered position out of contact with themultiplex cartridge, the magnet assemblies 552, 570 rotate downwardly totheir retracted positions away from the multiplex cartridge, the mixingmotor assembly 700 is pivoted outwardly out of an engagement with themultiplex cartridge, and the multiplex cartridge latch 654 is pivoted sothat the hook 656 disengages from the multiplex cartridge. Therefore,the multiplex cartridge is not contacted or otherwise engaged by any ofthe components of the multiplex cartridge processing assembly 470, andthe multiplex cartridge 10 can be ejected by the cartridge ejectorassembly 670.

Blister Compression Mechanism Assembly (Top Bay)

Details of a blister compression mechanism assembly 750 are shown inFIG. 51; which is an exploded prospective view of the blistercompression mechanism assembly 750. The assembly 750 comprises a cam armplate 752 and an array 754 of cam arm-operative compression mechanismsoperatively mounted within the cam arm plate 752. The cam arm plate 752is mounted on top of the blister plate 644 of the mounting plate 640.The compression mechanisms of the array 754 comprise compressionmechanisms configured to compress collapsible fluid compartments orblisters of the multiplex cartridge 10, compression mechanismsconfigured to compress lance blisters of the cartridge, and compressionmechanisms configured to press down on and close active valve assembliesof the cartridge. The various compression mechanisms of the array 754are aligned with blister holes 646 formed in the blister plate 644 sothat the compression mechanisms of the array 754 can access the blistersand active valves of the multiplex cartridge 10 positioned below theblister plate 644 within the processing bay 440.

In various embodiments, the LED PCB 466 is attached to the cam arm plate752.

The blister compression mechanism assembly 750 further includes a camfollower plate 820 mounted to the cam arm plate 752 for linear movementwith respect to the cam arm plate. In various embodiments, one edge ofthe cam follower plate 820 is secured to a linear guide rail 822attached to a top surface of the cam arm plate 752 by means of linearguide carriages 824 a and 824 b attached to the cam follower plate 820.An opposite edge of the cam follower plate 820 is secured againstvertical movement by a hold down element 826 (or Z-axis constraint)mounted within a recess 753 formed in the cam arm plate 752, e.g., bysuitable fasteners, and including a longitudinal slot 828 along one edgethereof which receives a stepped edge 830 of the cam follower plate 820.Suitable materials for construction of the hold down element includeDelrin and brass. Accordingly, the cam follower plate 820 is fixed inthe Z, or vertical direction or normal direction with respect to theplane of the cam arm plate 752, at a given space from the cam arm plate752 and is allowed movement in a longitudinal direction corresponding tothe longitudinal direction of the linear guide rail 822 and generallyparallel to the plane of the cam arm plate 752 but is restricted frommovement in any direction transverse to the linear guide rail 822.

Powered movement of the cam follower plate 820 with respect of the camarm plate 752 is effected by a cam follower plate motor 834 attached bymeans of a linear actuator 836 to a drive bracket 840 that is attachedto an edge of the cam follower plate 820. In various embodiments, themotor 834 further includes a rotary encoder 838 for providing precisecontrol of and feedback from the motor 834. In various embodiments, thedrive bracket 840 has an “L” shape with a first portion extending awayfrom an attachment point to the cam follower plate 820 in a planegenerally corresponding to the plane of the cam follower plate and asecond portion extending downwardly in a direction that is generallynormal to the plane of the cam follower plate. The linear actuator 836is attached to the drive bracket 840 at a lower end of the second,downwardly-extending portion of the drive bracket 840. Thisconfiguration of the drive bracket 840 limits the amount by which thecam follower plate motor 834 extends above the cam follower plate 820,to thus maintain a slim profile of the processing bay 440.

In various embodiments, a sensor mechanism is provided for indicatingwhen the cam follower plate 820 is in a particular, pre-defined positionwith respect to the cam arm plate 752. In one embodiment, the sensormechanism may comprise a home switch 842 that is mounted to the cam armplate 752 and is contacted by a home switch contact surface 832 of thecam follower plate 820 when the cam follower plate 820 has been moved toa home position relative to the cam arm plate 752.

In various embodiments, cam arm plate 752 includes two optical sensors810, 812 positioned so as to correspond spatially to the locations ofthe inlet and outlet optical ports 14, 16, respectively (see FIG. 1).Sensors 810, 812 are constructed and arranged to detect (e.g., generatea signal) fluid flow through inlet optical sensing chamber 154 andoutlet optical sensing chamber 158 of the sample preparation module 70(see, e.g., FIG. 15). Optical sensors 810, 812 may be connected to andat least partially controlled by the LED PCB 466.

Compression Mechanism

Details of the compression mechanisms are shown in FIGS. 52, 53 and 54.FIG. 52 is a bottom partial plan view of the cam arm plate 752 showingcompression pads of the array 754 of compression mechanisms. FIG. 53 isa top perspective view of the compression mechanisms of the array 754isolated from the cam arm plate 752. FIG. 54 is a bottom perspectiveview of the compression mechanisms of the array 754 isolated from thecam arm plate 752.

The array 754 comprises a plurality of fluid blister compressionmechanisms, each configured to, when actuated, apply a compressive forceonto an associated deformable fluid blister and thereby compress thedeformable blister. In the illustrated embodiment, there are five fluidblister compression mechanisms 756 a, 756 b, 756 c, 756 d, and 756 ecorresponding to the deformable fluid chambers 34 a, 36 a, 38 a, 40 a,and 42 a, respectively, of the multiplex cartridge.

The array 754 further includes a plurality of lance blister compressionmechanisms, each configured to, when actuated, apply a compressive forceonto an associated lance blister that is associated with one of thedeformable fluid blister and thereby compress the lance blister andlance the fluid seal within the lance blister. In the illustratedembodiment, there are five lance blister compression mechanisms 760 a,760 b, 760 c, 760 d, and 760 e corresponding to the lance blisters 34 b,36 b, 38 b, 40 b, and 42 b, respectively, of the multiplex cartridge.

The array 754 further includes a compression mechanism 758 havingsubstantially the same configuration as a lance blister compressionmechanism 760 a-e and corresponding to blister 44 of the multiplexcartridge.

The array 754 includes two valve actuator compression mechanisms 762 a,762 b associated with sample valve assembly 204 and waste valve assembly219, respectively (see FIG. 15). Each of the valve actuator compressionmechanisms 762 a, 762 b is configured to, when actuated, apply acompressive force on the valve actuator tabs 20, 18 (see FIG. 1),respectively, and thus to actuate, and close, the active valves 219 and204.

Details of the constructions of each of the various compressionmechanisms are shown in FIGS. 53 and 54, as well as in FIGS. 55A, 55B,and 55C. FIG. 55A is an exploded perspective view of a single fluidblister compression mechanism. FIG. 55B is an exploded prospective viewof a single lance blister compression mechanism. FIG. 55C is an explodedperspective view a valve actuator compression mechanism.

The blister compression mechanism assembly employs principles andconcepts described in U.S. patent application Ser. No. 14/206,817entitled “Apparatus and Methods for manipulating deformable fluidvessels” the contents of which are hereby incorporated by reference. Inparticular, the blister compression mechanism assembly is constructedand arranged to convert the horizontal movement of cam follower pate 820into vertical, or partially vertical, movement of the compressionmechanisms to compress a fluid blister, a lance blister, and a valveassembly without requiring pneumatic, electromechanical, or othercomponents at larger distances above and/or below the multiplexcartridge 10 to thus maintain a slim profile of the processing bay 440.

Referring to FIG. 55A, each fluid blister compression mechanism, such asthe fluid blister compression mechanism 756 a, includes a cam arm 764with a cam surface 766 formed along a top edge thereof. The cam arm 764is mounted within the cam arm plate 752 for pivoting movement about anarm pivot pin 768 extending through a hole formed in one end of the camarm 764. The cam arm 764 is disposed within a slot 765 formed in the camarm plate 752, and the arm pivot pin 768 is mounted within the cam armplate 752 transversely to that slot (see FIG. 52). A compression pad 772is pivotally mounted to an opposite end of the cam arm 764 for pivotingmovement about a pad pivot pin 774 extending through a hole formed inthe opposite end of the cam arm 764. In various embodiments, thecompression pad 772 is disposed within a blind recess 773 formed in abottom surface of the cam arm plate 752 in a shape generally conformingto the shape of the compression pad 772 (see FIG. 52).

The fluid blister compression mechanism 756 a is configured to pivotwith respect to the cam arm plate 752 about the arm pivot pin 768between a retracted position in which the compression mechanism is notapplying pressure to the associated fluid blister and an extended, ordeployed, position in which the compression mechanism is applying acompressive force onto the fluid blister. A torsion spring 770 biasesthe compression mechanism 756 a into the retracted position. In theretracted position, the cam arm 764 is substantially disposed within thecorresponding slot 765 formed in the cam arm plate 752 and thecompression pad 772 is disposed within the pad recess 773 formed in thecam arm plate 752 so that the blister-contacting surface of thecompression pad 772 is substantially flush with a surface of the cam armplate 752. In the extended position, the cam arm 756 is rotated aboutthe cam arm pivot pin 768 so that the compression pad 772 is extendedbeneath the cam arm plate 752 to compress and collapse the reagentblister disposed beneath the compression pad 772.

The cam surface 766 may include a convex bulge, or other feature, that,in various embodiments, extends above a top surface of the cam arm plate752 (see FIG. 51, showing cam features of the cam arms of the array 754of compression mechanisms extending above cam arm plate 752). When thecam surface 766 is engaged by a cam follower element moving relative tothe cam arm 764 over the cam surface 766, the cam arm 764 is caused topivot from the retracted position to the extended position as the camfollower moves over the convex bulge of the cam surface 766. As the camfollower element moves off the cam surface 766, the cam arm 764 returnsto the retracted position under the force of the torsion spring 770.

The cam arm 764 is preferably made from a material having sufficientstrength to withstand forces applied to it by a cam follower elementpushing the cam arm 764 against a collapsible fluid blister and havingsuitable machinability. Suitable materials include steel forapplications in which the cam follower element comprises a roller thatrolls over the cam surface 766. For applications in which the camfollower element comprises a sliding (i.e., non-rolling) element thatslides over the cam surface 766, suitable materials include lowfriction, low abrasion materials, such as nylon or alubricant-impregnated material, such as oil-impregnated bronze.

In various embodiments, the construction and operation of the otherfluid blister compression mechanisms, 756 b, 756 c, 756 d, and 756 e aresubstantially the same as that of the fluid blister compressionmechanism 756 a, although the size and shape of the compression pads(e.g., compression pad 772) may vary from one fluid blister compressionmechanism to the next according to the size and shape of the fluidblister that is to be compressed by the compression mechanism.

Referring to FIG. 55B, each lance blister compression mechanism, such asthe lance blister compression mechanism 760 a, includes a cam arm 780with a cam surface 782 formed along a top edge thereof. The cam arm 780is mounted within the cam arm plate 752 for pivoting movement about anarm pivot pin 784 extending through a hole formed in one end of the camarm 780. The cam arm 780 is disposed within a slot 781 formed in the camarm plate 752, and the arm pivot pin 784 is mounted within the cam armplate 752 transversely to that slot (see FIG. 52). A compression pad 788is formed or positioned on an opposite end of the cam arm 780. Invarious embodiments, the compression pad 788 is disposed within a blindrecess 789 formed in a bottom surface of the cam arm plate 752 in ashape generally conforming to the shape of the compression pad 788 (seeFIG. 52).

The lance blister compression mechanism 760 a is configured to pivotwith respect to the cam arm plate 752 about the arm pivot pin 784between a retracted position in which the compression mechanism is notapplying pressure to the associated lance blister and an extended, ordeployed, position in which the compression mechanism is applying acompressive force onto the lance blister. A torsion spring 786 biasesthe compression mechanism 760 a into the retracted position. In theretracted position, the cam arm 780 is substantially disposed within thecorresponding slot 781 formed in the cam arm plate 752 and thecompression pad 788 is disposed within the pad recess 789 formed in thecam arm plate 752 so that the blister-contacting surface of thecompression pad 788 is substantially flush with a surface of the cam armplate 752. In the extended position, the cam arm 780 is rotated aboutthe cam arm pivot pin 784 so that the compression pad 788 is extendedbeneath the cam arm plate 752 to compress and collapse the lance blisterdisposed beneath the compression pad 788.

The cam surface 782 may include a convex bulge, or other feature, that,in various embodiments, extends above a top surface of the cam arm plate752 (see FIG. 51, showing cam features of the cam arms of the array 754of compression mechanisms extending above the cam arm plate 752). Whenthe cam surface 782 is engaged by a cam follower element moving relativeto the cam arm 780 over the cam surface 782, the cam arm 780 is causedto pivot from the retracted position to the extended position as the camfollower moves over the convex bulge of the cam surface 782. As the camfollower element moves off the cam surface 782, the cam arm 780 returnsto the retracted position under the force of the torsional spring 786.

The cam arm 780 is preferably made from a material having sufficientstrength to withstand forces applied to it by a cam follower elementpushing the cam arm 780 against a collapsible lance blister and havingsuitable machinability. Suitable materials include steel forapplications in which the cam follower element comprises a roller thatrolls over the cam surface 782. For applications in which the camfollower element comprises a sliding (i.e., non-rolling) element thatslides over the cam surface 782, suitable materials include lowfriction, low abrasion materials, such as nylon or alubricant-impregnated material, such as oil-impregnated bronze.

In various embodiments, the construction and operation of the otherlance blister compression mechanisms, 760 b, 760 c, 760 d, and 760 e,and the compression mechanism 758, are substantially the same as that ofthe lance blister compression mechanism 760 a.

Referring to FIG. 55C, each valve actuator compression mechanism, suchas valve actuator compression mechanism 762 a, includes a cam arm 790with a cam surface 792 formed along a top edge thereof. The cam arm 790is mounted within the cam arm plate 752 for pivoting movement about anarm pivot pin 794 extending through a hole formed in one end of the camarm 790. The cam arm 790 is disposed within a slot 791 formed in the camarm plate 752, and the arm pivot pin 794 is mounted within the cam armplate 752 transversely to that slot (See FIG. 52). A contact pad 798 isformed or positioned on an opposite end of the cam arm 790. In variousembodiments, the contact pad 798 is disposed within a blind recess 799formed in a bottom surface of the cam arm plate 752 in a shape generallyconforming to the shape of the contact pad 798 (see FIG. 52).

In various embodiments, the contact pad 798 may further include acontact pin, or point, 800 projecting from the contact pad 798. Thecontact point is configured to engage a small dimple or depressionformed in the top surface of the valve actuator tab 18 or 20 when thevalve actuator compression mechanism is pressing against the tab toprevent the compression mechanism from slipping off the valve actuatortab. Also, in various embodiments, a portion of the contact pad 798, andthe contact pin 800, may be offset from the cam arm 690 to accommodatespace and orientation limitations within the array 754 of compressionmechanisms.

The valve actuator compression mechanism 762 a is configured to pivotwith respect to the cam arm plate 752 about the arm pivot pin 794between a retracted position in which the compression mechanism is notapplying pressure to the associated valve actuator tab and active valveassembly and an extended, or deployed, position in which the compressionmechanism is applying a compressive force onto the actuator tab andvalve assembly. A torsion spring 796 biases the compression mechanism762 a into the retracted position. In the retracted position, the camarm 790 is substantially disposed within the corresponding slot 791formed in the cam arm plate 752 and the contact pad 798 is disposedwithin the pad recess 799 formed in the cam arm plate 752 so that thecontact surface of the contact pad 798 is substantially flush with asurface of the cam arm plate 752. In the extended position, the cam arm790 is rotated about the cam arm pivot pin 794 so that the contact pad798 is extended beneath the cam arm plate 752 to deflect the valveactuator tab downwardly and close the associated valve assembly disposedbeneath the valve actuator tab.

The cam surface 792 may include a convex bulge, or other feature, that,in various embodiments, extends above a top surface of the cam arm plate752 (see FIG. 51, showing cam features of the cam arms of the array 754of compression mechanisms extending above the cam arm plate 752). Whenthe cam surface 792 is engaged by a cam follower element moving relativeto the cam arm 790 over the cam surface 792, the cam arm 790 is causedto pivot from the retracted position to the extended position as the camfollower moves over the convex bulge of the cam surface 792. As the camfollower element moves off the cam surface 982, the cam arm 790 returnsto the retracted position under the force of the torsion spring 796.

The cam arm 790 is preferably made from a material having sufficientstrength to withstand forces applied to it by a cam follower elementpushing the cam arm 790 against a valve assembly and having suitablemachinability. Suitable materials include steel for applications inwhich the cam follower element comprises a roller that rolls over thecam surface 792. For applications in which the cam follower elementcomprises a sliding (i.e., non-rolling) element that slides over the camsurface 792, suitable materials include low friction, low abrasionmaterials, such as nylon or a lubricant-impregnated material, such asoil-impregnated bronze.

In various embodiments, the construction and operation of the othervalve actuator compression mechanism 762 b are substantially the same asthat of the valve actuator compression mechanism 762 a.

Details of the cam follower plate 820 are shown in FIGS. 56 and 57. FIG.56 is a bottom plain view of the cam follower plate 820, and FIG. 57 isa bottom perspective view of the cam follower plate 820.

The cam follower plate 820 includes a number of generally parallel,longitudinal cam grooves 850, 852, 854, 856, 858 and 860. Each of thegrooves 850-860 of the cam follower plate 820 receives a portion of oneor more the cam arms 764, 780, 790 of the compression mechanisms of thearray 754. In addition, each groove 850-860 includes one or more camfollower elements, e.g., in the form of ribs or rollers formed orpositioned at discreet positions along the corresponding groove.

The cam follower plate 820, as noted above, is configured for linearmovement relative to the cam arm plate 752 in a plane that is parallelto the cam arm plate 752. As the cam follower plate 820 moves relativeto the cam arm plate 752, when a cam follower element within a camgroove encounters the cam surface of the cam arm of the compressionmechanism (e.g., cam surface 766, 782, or 792 of cam arms 764, 780, or790, respectively), the cam arm is pushed downwardly, pivoting about itsrespective arm pivot pin (e.g., pivot pin 768, 784, or 794) to cause thecompression mechanism to compress the blister (e.g., compressible fluidblister or lance blister) or press the active valve assembly disposedbeneath that compression mechanism.

During movement of the cam follower plate 820 with respect to the camarm plate 852, the relative locations of the compression mechanisms ofthe array 754 of compression mechanisms and the cam follower ribs formedin the grooves 850, 852, 854, 856, 858, and 860 define the sequence inwhich the compression mechanisms are actuated.

Software and Hardware

As generally and specifically describe above, aspects of the disclosureare implemented via control and computing hardware components,user-created software, data input components, and data outputcomponents. Hardware components include computing and control modules(e.g., system controller(s)), such as microprocessors and computers,configured to effect computational and/or control steps by receiving oneor more input values, executing one or more algorithms stored onnon-transitory machine-readable media (e.g., software) that provideinstruction for manipulating or otherwise acting on the input values,and output one or more output values. Such outputs may be displayed orotherwise indicated to a user for providing information to the user, forexample information as to the status of the instrument or a processbeing performed thereby, or such outputs may comprise inputs to otherprocesses and/or control algorithms. Data input components compriseelements by which data is input for use by the control and computinghardware components. Such data inputs may comprise positions sensors,motor encoders, as well as manual input elements, such as graphic userinterfaces, keyboards, touch screens, microphones, switches,manually-operated scanners, voice-activated input, etc. Data outputcomponents may comprise hard drives or other storage media, graphic userinterfaces, monitors, printers, indicator lights, or audible signalelements (e.g., buzzer, horn, bell, etc.).

Software comprises instructions stored on non-transitorycomputer-readable media which, when executed by the control andcomputing hardware, cause the control and computing hardware to performone or more automated or semi-automated processes.

Sample Preparation Process

An exemplary sample preparation process that may be performed in thesample preparation module 70 is described and illustrated in FIGS.16-23. Persons of ordinary skill will recognize that sample preparationprocesses other than that described herein—e.g., reordering of the stepsfrom what is described herein, the omission of certain steps describedherein, and/or addition of certain steps—may be performed with thesample preparation module or a modified version of the samplepreparation module.

In a first step, illustrated in FIG. 16, a fluid sample specimen isdispensed into the sample well 78. In general, the multiplex cartridge10 is designed to process liquid or solid samples. Liquid samples mayinclude blood, serum, plasma, urine, saliva, cerebral spinal fluid,lymph, perspiration, semen or epithelial samples such as cheek,nasopharyngeal, anal or vaginal swabs to which lysis buffer has beenadded to resuspend the cells. Solid samples, such as feces or tissuesamples (e.g. tumor biopsies), generally need to be resuspended anddiluted in a buffer, e.g., the Cary Blair transport medium. The samplewell 78 may then be closed using the sample cap 84 (see FIG. 6), and themultiplex cartridge 10 is then placed in a processing instrument (e.g.,into the processing bay 440 of the processing module 410 of theinstrument 400).

In a first step performed within the instrument, as illustrated in FIG.17, the lance blister 34 b associated with the deformable compartment 34a is compressed by an external actuator (e.g., the compression mechanism760 a) to press a bead or other opening device through a closing seal(i.e., lance the seal with the bead or other device), and then thedeformable compartment 34 a is compressed by an external actuator (e.g.,the compression mechanism 756 a) to force a process fluid containedtherein into the first inlet port 136 formed in the substrate 72. In oneembodiment, the process fluid contained in the deformable compartment 34a is a lysis buffer. The fluid is directed by the first fluid channel150 from the inlet port 136 to the sample well 78, where the fluidenters the sample well 78 through the inlet snorkel 80. In addition, anexternal pump (e.g., pump 458) connected to the sample preparationmodule 70 at the pump port 104 generates pressure that is applied to thecontents of the sample well 78 via the pressure conduit 106.

The pressure generated by compressing the deformable compartment 34 aand the pressure applied at pressure conduit 106 pushes the fluidcontents—comprising the fluid sample and the contents of the deformablecompartment 34 a—from the sample well 78 through the second fluidchannel 152 to the lysis chamber inlet 122. The fluid continues to flowthrough the lysis chamber, exiting the outlet 124, where it is directedby the third fluid channel 156 and a portion of the fifth fluid channel162 into the mixing well 90. As the fluid stream first enters or exitsthe lysis chamber 120 and passes through the inlet optical sensingchamber 154 or the outlet optical sensor chamber 158, it is detectedthrough the associated optical port 14 or 16 formed in the upper shroud12 (see FIG. 1) by an optical detector (e.g., optical detector(s)mounted in LED PCB 466). A signal from the optical detector indicatingfluid flow (e.g., an air-fluid interface) through the inlet or outletoptical sensing chamber 154 or 158 activates the motor 128 of the lysischamber mixer to disrupt the fluid flowing through the lysis chamber 120with lysis beads contained within the lysis chamber 120. The motor 128continues to operate until a signal from an optical detector indicatingthe end fluid flow through the inlet or outlet optical sensing chamber154 or 158—and thus the end of flow through the lysis chamber 120,deactivates the motor 128.

As the fluid mixture is flowing into the mix compartment 90, the passivevalve port 108 remains open so that pressure within the mixing well 90does not rise to a level that will open the passive valve assembly 220.Thus, at the conclusion of the step illustrated in FIG. 17, the mixingwell 90 will contain a mixture of fluid sample and the contents of thedeformable compartment 34 a (e.g., a lysis buffer) which has beenphysically lysed by the lysis mixer and lysis beads contained in thelysis chamber 120.

Referring now to FIG. 18, after the step shown in FIG. 17, the pneumaticpump applying pressure at pressure port 104 is turned off, e.g., after aprescribed period of operation, and the third deformable compartment 44is compressed by an external actuator (e.g., the compression mechanism758) to force the contents of the deformable compartment 44 into thethird inlet port 140. In one embodiment, the contents of the deformablecompartment 44 comprise magnetic target capture beads.

Next, the lance blister 36 b associated with the deformable compartment36 a is compressed by an external actuator (e.g., the compressionmechanism 760 e) to press a bead or other opening device through aclosing seal (i.e., lance the seal with the bead or other device), andthen the deformable compartment 36 a is compressed by an externalactuator (e.g., the compression mechanism 756 e) to force a processfluid contained therein into the second inlet port 138 formed in thesubstrate 72. The process fluid then flows through the fourth fluidchannel 160 and the fifth fluid channel 162 to the mixing well 90. Thecontents of the deformable compartment 36 a may comprise a bindingbuffer for facilitating the binding of the target capture beads to thetarget analyte(s). The flowing fluid past the third inlet port 140,under the pressure generated by the compression of the deformablecompartment 36 a, transports the fluid contents of the deformablecompartment 36 a and the contents of the deformable compartment 44through the fifth fluid channel 162 to the mixing well 90.

As noted above, in an alternate embodiment, the magnetic beads may beprovided in the form of a lyophilized pellet contained within the mixingwell 90, and the deformable compartment 44, the associated externalactuator (e.g., the compression mechanism 758), and the step of burstingthe deformable compartment 44 may be omitted.

After the step illustrated in FIG. 18 is completed, the rotary mixer 192within the mixing well 90 may be activated (e.g., by the mixing motorassembly 700) to stir the contents of the mixing well 90. In variousembodiments, a lyophilized or other dried reagent form may bepre-positioned in the mixing well 90 and is dissolved or reconstitutedby the fluids transported into the mixing well 90. The rotary mixer 192helps facilitate the dissolution or reconstitution of the dried reagentand mixes all the materials contained in the mixing well to form ahomogeneous fluid mixture.

Referring to FIG. 19, a next step comprises collapsing the lance blister38 b (e.g., with the compression mechanism 760 b) associated with thedeformable compartment 38 a to thereby open the compartment to thefourth inlet port 142. The deformable compartment 38 a is then collapsed(e.g., with the compression mechanism 756 b to direct the fluid contentsthereof into the fourth inlet port 142, through the sixth fluid channel164 and to the first outlet port 182, where the fluid exits the samplepreparation module 70. The first outlet port 182 is in communicationwith the inlet port 252 of the reaction module 240 as explained above.The fluid contained in the deformable compartment 38 a may comprise animmiscible fluid, e.g., an oil, which is used to fill a reaction space295 within the reaction module 240 between the top plate 241 and thefluidic processing panel 354, as shown in FIG. 30.

Referring now to FIG. 20, the lance blister 40 b associated with thedeformable compartment 40 a is collapsed by an external actuator (e.g.,the compression mechanism 760 c) to open the compartment to the fifthinlet port 144, and then the deformable compartment 40 a is collapsed byan external actuator (e.g., the compression mechanism 756 c) to forcethe fluid contents thereof into the fifth inlet port 144. The fluidcontents flow from the fifth inlet port 144 to a second outlet 188 via aseventh channel fluid 166. In one embodiment, the fluid content of thedeformable compartment 40 a comprises a rehydration or elution bufferthat flows from the second exit port 188 into the rehydration buffercompartment 276 of the reaction module 240 via inlet 278, as shown inFIG. 31 and described above. The same buffer solution contained in thedeformable compartment 40 a may be used for both rehydration of dried orlyophilized reagents or other substances or for elution of nucleic acidor other target analyte from a substrate with which it is bound.

Referring now to FIG. 21, in a next step, the active valve assembly 204is closed by an external actuator (e.g., the valve actuator 762 b)pressing down on the valve. The pneumatic pump coupled to the pump port104 is activated to pressurize the mixing well 90 via the pressureconduit 106, a portion of the first fluid channel 150, the second fluidchannel 152, the third fluid channel 156, and a portion of the fifthfluid channel 162. At the same time, the passive valve port 108 isclosed to allow a pressure buildup in the mixing well 90 that willactuate the passive valve assembly 220, thereby opening the passivevalve 220 to allow fluid contents of the mixing well 90 to flow, via thechannels 92 and 172, through the capture compartment 100. Fluid flowingthrough the capture compartment 100 flows through the thirteenth fluidchannel 178, but is prevented by the closed active valve assembly 204from flowing into the fourteenth fluid channel 180. The active valveassembly 219 remains open so that fluid within the thirteenth fluidchannel 178 flows into the tenth fluid channel 172 and into the wastechamber 102. While the fluid is flowing through the capture compartment100, the contents are subjected to a magnetic force, for example, byplacement of an external magnet (e.g., by deploying the samplepreparation magnet assembly 570) in proximity to the capture compartment100. The magnetic force retains magnetic target capture beads and targetanalyte(s) (e.g., nucleic acid(s)) bound thereto within the capturecompartment 100 while the remainder of the contents of the mixing well90 flows through the capture compartment 100 and into the waste chamber102.

Referring now to FIG. 22, in a next step, the valve assembly 204 remainsclosed and the valve assembly 219 remains open, and the lance blister 42b associated with the deformable compartment 42 a is collapsed (e.g.,with compression mechanism 760 d) to thereby open the compartment to thesixth inlet port 146. The deformable compartment 42 a is then partiallycollapsed (e.g., with the compression mechanism 756 d) to dispense aportion (e.g., approximately 50%) of its contents into the sixth inletport 146. In one embodiment, the fluid contents of the deformablecompartment 42 a comprise a wash buffer which flows from the sixth inletport 146 via the twelfth fluid channel 176 to the capture compartment100. The wash fluid flows over the capture beads that are immobilized(e.g., by a magnet) within the capture compartment 100 and flows throughthe channels 178, 172, and 174 to the waste chamber 102 to thereby carryunbound material and other debris into the waste chamber 102.

Referring now to FIG. 23, in a next step, the waste valve assembly 219is closed by an external actuator (e.g., the valve actuator 762 a), andthe sample valve assembly 204 is opened by removing the externalactuator. Next, the remainder of the deformable compartment 42 a iscollapsed by the external actuator (e.g., the compression mechanism 756d), thereby forcing the remainder of the fluid (e.g., a wash buffer)through the twelfth fluid channel 176 into the capture compartment 100.The magnetic force is removed from the capture compartment 100 (e.g., byretracting the sample preparation magnet assembly 570) so that themagnetic beads within the capture compartment 100 are released and canbe carried by the fluid flowing through the capture compartment 100through the thirteenth fluid channel 178 through the sample valveassembly 204 and the fourteenth fluid channel 180 to a third outlet 190.The fluid flowing from the third outlet 190, which now comprises an atleast partially purified target analyte carried on the magnetic beads,is dispensed into the sample compartment 266 of the reaction module 240via the inlet 268, as shown in FIG. 31 and described above.

In FIGS. 56 and 57, each of the cam follower ribs formed in the camgrooves 850-860 of the cam follower plate 820 is indicated by a uniqueparenthetical number (1)-(14). As the cam follower plate 820 is movedrelative to the cam arm plate 852 in the direction “A,” the cam followerribs formed in various cam grooves 850-860 contact the compressionmechanisms of the actuator array 754 in a predetermined sequence so asto open the various reagent chambers and dispense their contents andactuate the various active values in a specified sequence. Theparenthetical numbers assigned to the cam follower ribs in FIGS. 56, 57indicates the sequence in which each rib contacts an associated cam armof the compression mechanisms of the array 754 to actuate thecompression mechanisms in a sequence corresponding to the samplepreparation process performed in the sample preparation module 70 asdescribed above and shown in FIGS. 16-23. The table below showscorrespondence between each cam follower rib of the cam follower plate820, the process step, the corresponding compression mechanism, andcompressing collapsible chamber or active valve of the multiplexcartridge 10 for the process shown in FIGS. 16-23.

Compression Compressible Follower Mechanism/ Chamber/ Element ProcessStep Valve Actuator Active Valve (1) Open Lysis Lance Blister 760a 34b (2)* Open and dispense 758  44  magnetic beads (3) Dispense Lysisbuffer 756a 34a (4) Open Binding Buffer 760e 36b Lance Blister (5)Dispense Binding Buffer 756e 36a (6) Open Oil Lance Blister 760b 38b (7)Dispense Oil 756b 38a (8) Open Elution/Reconstitu- 760c 40b tion LanceBlister (9) Dispense Elution/ 756c 40a Reconstitution Buffer (10)  Closesample Valve 762b 204 assembly (11)  Open Wash Buffer Lance 760d 42bBlister (12)  Dispense 50% wash buffer 756d 42a (13)  Close waste valveassembly 762a 219 (14)  Dispense 100% wash buffer 756d 42a *step (2) isoptional and may be omitted if magnetic beads are provided directly,e.g., by a lyophilized pellet, in the mixing well 90.

Sample Reaction Process

The sample material that is dispensed from the sample processing module70 into the sample compartment 266 of the reaction module 268 issubjected to a reaction process with the reaction module 240. In oneexemplary embodiment, that reaction process includes PCR amplificationand analyte detection.

An exemplary process will be described with reference to flow chart 900in FIG. 60. Although the various elements (steps) of flow chart 900 inFIG. 60 are shown as sequential steps having a prescribed order, itshould be understood that the process 900 as illustrated is exemplaryand not intended to be limiting. Persons of ordinary skill willrecognize that many of the various elements (steps) of the process 900can be performed in different orders than are shown and describedherein, can be performed simultaneously or substantially simultaneouslywith other elements (steps), or can be omitted altogether. Thus, theorder of the elements (steps) as shown in FIG. 60 should not be viewedas limiting unless a specific order for two or more elements (steps) isspecifically prescribed or otherwise suggested by the context of thedescription (e.g., a mixture must first be formed before that mixturecan be incubated or otherwise manipulated).

In step S1, an aliquot of the elution/reconstitution buffer (e.g., 15μl) is dispensed by electrowetting droplet manipulation from therehydration buffer zone 372 (FIG. 59) (and rehydration buffercompartment 376 of top plate 241 (FIGS. 26, 27)) to an electrowettingpathway defining the exonuclease zone 384 (FIG. 59).

As noted above, in an embodiment of the invention, the region of thereaction module 240 between the top plate 241 and the fluidic processingpanel 354 may be filled with a process fluid, such as an immisciblefluid such as oil, and the droplets are manipulated through the oil.

In step S2, an aliquot of the sample mixture (comprising magnetic beadswith DNA material bound thereto and wash solution from the samplepreparation module 70) is retained by electrowetting manipulation withinthe sample bead zone 368 (FIG. 59) (and the sample compartment 266 ofthe top plate 241 (FIGS. 26, 27)), while the magnetic beads are pulledout of the aqueous solution held within the sample bead zone 368 by amagnet that is focused on position 369 (referred to as the beadcollection area). The bead collection area 369 corresponds to theposition of the focusing magnet 558 of the cartridge magnet assembly 552(See FIG. 49B) adjacent to the fluid processing panel 554 of themultiplex cartridge 10 when the cartridge magnet assembly 552 is in thedeployed position. During the process of collecting the magnetic beadsat the bead collection area 369, the aqueous solution may be movedthroughout the sample bead zone 368 by selective activation of differentelectrowetting pads to move the aqueous droplets containing the magnetbeads to positions in closer proximity to the magnetic force at the beadcollection area 369.

In Step S3, sample waste (i.e., wash buffer and other materials fromwhich the magnetic beads have been removed in Step S2), is retained byelectrowetting droplet manipulation within the sample bead zone 368 (andthe sample compartment 266), thereby separating the magnet beads, andthe target analyte material bound thereto, from the other constituentsubstances of the sample bead mixture that was delivered from the samplepreparation module 70 to the sample bead zone 368.

In Step S4, an amount of the reconstitution buffer that was dispensedfrom the rehydration buffer zone 372 in Step S1 may be moved byelectrowetting droplet manipulation to the PCR reagent zone 376 (FIG.59) (and the buffer compartment 296 of the top plate 241 (FIGS. 26,27)). Resuspension of the dried PCR reagent contained within the PCRreagent zone 376 occurs by oscillating movements of the droplets betweenthe electrowetting pads within the PCR reagent zone 376.

In Step S5, an amount of the reconstitution buffer that was dispensedfrom the rehydration buffer zone 372 and which was not transported tothe PCR reagent zone 376 is transported by electrowetting dropletmanipulation over the magnetic beads held by the magnetic force at thebead collection area 369 for a final bead wash. After the final beadwash, the reconstitution buffer is then moved by electrowetting dropletmanipulation to an end of the center pathway corresponding to theexonuclease zone 384 where it is held by electrowetting dropletmanipulation apart from the magnetic beads held at the bead collectionarea 369.

In the Step S6, the reconstituted PCR buffer within the PCR reagent zone376 is distributed by electrowetting droplet manipulation to the primercocktail positions of each of the thermal cycling tracks 364 a, 364 b,364 c, and 364 d. One primer cocktail position 366 a at a proximal endof the thermal cycling track 364 d is labeled in FIG. 59. Each of theother thermal cycling tracks 364 a, 364 b, and 364 c has a similarprimer cocktail location. The combination of reconstituted PCR reagentwith the dried primer cocktail at the primer cocktail position (e.g.,position 366) reconstitutes the primer cocktail at that position. Inthis configuration, the reaction module 240 is configured to perform onePCR reaction in each of the thermal cycling tracks 364 a, 364 b, 364 c,and 364 d.

In an alternate embodiment, a primer cocktail may also be provided atthe distal end of each thermal cycling track 364 a, 364 b, 364 c, and364 d. One primer cocktail position 366 b at a distal end of thermalcycling track 364 d is labeled in FIG. 59. Each of the other thermalcycling tracks 364 a, 364 b, and 364 c may have a similar primercocktail location. In such a configuration, the reaction module 240 isconfigured to perform two PCR reactions in each of the thermal cyclingtracks 364 a, 364 b, 364 c, and 364 d.

In Step S7, the magnetic force is removed from the bead collection area369 (e.g., by moving the cartridge magnet assembly 552 to its retractedposition). Reconstitution/elution buffer is moved by electrowettingdroplet manipulation from the central pathway 384 to the bead collectionarea 369, and a mixture of the magnetic beads and reconstitution/elutionbuffer from the rehydration buffer zone 372 is shuttled back and forthalong the path 384 by electrowetting droplet manipulation to elute theDNA material (or other target analyte) from the magnetic beads.

After a sufficient elution period, in Step S8, the cartridge magnetassembly 552 is again deployed to apply a magnetic force (via thefocusing magnet 558) to the bead collection area 369 to attract andretain (immobilize) the magnetic beads from which the DNA material hasbeen eluted, and the eluted DNA material is transferred byelectrowetting droplet manipulation to a PCR staging area at a proximalend of each of the thermal cycling tracks 364 a, 364 b, 364 c, and 364d. In the embodiment and orientation shown in FIG. 59, the PCR stagingarea is at the left end of thermal cycling tracks 364 a, 364 b, 364 c,and 364 d.

In Step S9, PCR droplets—comprising the eluted DNA material, thereconstituted PCR reagent, and the reconstituted PCR primer—are formedby electrowetting droplet manipulation at the PCR staging area of eachof the thermal cycling tracks 364 a, 364 b, 364 c, and 364 d. Each PCRdroplet is moved into a corresponding one of the thermal cycling tracks364 a, 364 b, 364 c, and 364 d, and a PCR process is performed byshuttling the droplets between two of the PCR (thermal cycling) regions382 a (at about, e.g., 60° C. for annealing and extension) and 382 b (atabout, e.g., 95° C. for denaturation) or 382 c (at about 60° C. forannealing and extension) and 382 b (at about, e.g., 95° C. fordenaturation). In another embodiment, two PCR droplets are transportedinto each thermal cycling track 364 a, 364 b, 364 c, and 364 d, and onedroplet is shuttled between heater areas 382 c and 382 b, whereas theother droplet is shuttled between heater areas 382 a and 382 b. The PCRprocess may last for about 40 minutes or less.

In Step S10, an amount of elution/reconstitution buffer is dispensed byelectrowetting droplet manipulation from the rehydration buffer zone 372and is transported by electrowetting droplet manipulation to theexonuclease reagent zone 374 (FIG. 59) (and the second buffercompartment 300 of the top plate 241 (FIG. 26, 27)) for resuspension ofthe dried exonuclease reagent. Resuspension of the dried exonucleasereagent contained within the exonuclease reagent zone 374 occurs byoscillating movements of the droplets between the electrowetting padswithin the exonuclease reagent zone 374. The reconstituted exonucleasereagent is then transported by electrowetting droplet manipulation fromthe exonuclease reagent zone 374 to PCR staging areas of the thermalcycling track 364 a, 364 b, 364 c, and 364 d.

In Step S11, following PCR (Step 9), each droplet that has gone throughthe PCR process is combined with an amount of the exonuclease agentresuspended in Step S10, transported by electrowetting dropletmanipulation to the exonuclease zone 384, and held in a separatelocation within the exonuclease zone 384. In various embodiments, anamount of elution/reconstitution buffer from the buffer zone 372 isadded to each PCR droplet by electrowetting droplet manipulation tobring the total volume of each droplet up to a preferred amount.

In Step S12, the droplet mixtures formed in Step S11, comprising the PCRproducts and the reconstituted exonuclease reagent, are then incubatedwithin the exonuclease region 380 and the exonuclease zone 384 at aprescribed temperature and for a prescribed period of time.

In Step S13, detection reagent within the hybridization zone 370 (FIG.59) (and the detection buffer compartment 280 of the top plate 241(FIGS. 26, 27)) is reconstituted with an amount of rehydration bufferfrom the rehydration buffer zone 372. In one embodiment, an amount ofrehydration buffer from the rehydration buffer zone 372 is moved viaelectrowetting droplet manipulation through the connecting passage 274(FIGS. 26, 27) between the detection buffer compartment 280 and therehydration buffer compartment 276.

In Step S14, an amount of the reconstituted detection reagent (e.g. 25μl) from the hybridization zone 370 is combined by electrowettingdroplet manipulation with each of the PCR droplets. Each PCR droplet isthen combined with a signal probe cocktail stored at positions 362 a,362 b, 362 c, and 362 d of the fluid processing panel 354. To effectmixing of the PCR droplet and the signal probe cocktail, and toresuspend the signal probe cocktail, each droplets may be transported byelectrowetting droplet manipulation around or within one of thedetection mixing zones 385 a, 385 b, 385 c, and 385 d.

In Step S15, the droplets are transported by electrowetting manipulationto the electrosensor arrays 363 a, 363 b, 363 c, and 363 d, where theyare subjected to further incubation within the detection region 378 andvarious analytes of interest are detected by electrosensing techniques,such as described above and/or described in publications incorporated byreference above.

Exemplary Embodiments

The following embodiments are encompassed by the foregoing disclosure.

Embodiment 1. A fluid sample processing cartridge comprising:

-   -   a substrate;    -   a sample well formed in the substrate and configured to receive        a volume of fluid sample;    -   a closure configured to be selectively placed over the sample        well;    -   a deformable fluid chamber supported on the substrate and        configured to hold a fluid therein when in an undeformed state        and to collapse upon application of an external compression        force to expel at least a portion of the fluid from the fluid        chamber, the deformable fluid chamber being in fluid        communication with the sample well via a channel formed in the        substrate;    -   a mixing well formed in the substrate, the mixing well being in        fluid communication with the sample well via a channel formed in        the substrate, the mixing well comprising a first peripheral        wall and a first floor defining a well and a fluid inlet snorkel        extending up a side of the first peripheral wall extending from        the channel communicating the mixing well to the sample well and        terminating below a top edge of the first peripheral wall; and    -   a driven mixing apparatus disposed within the mixing well and        constructed and arranged to mix the contents of the mixing well.

Embodiment 2. The fluid sample processing cartridge of embodiment 1,wherein the fluid inlet snorkel extends up an outer surface of the firstperipheral wall and terminates at an opening formed in the firstperipheral wall.

Embodiment 3. The fluid sample processing cartridge of embodiment 1 orembodiment 2, wherein the sample well comprising a second peripheralwall and a second floor defining a well and a fluid inlet snorkelextending up a side of the second peripheral wall and terminating belowa top edge of the second peripheral wall.

Embodiment 4. The fluid sample processing cartridge of any one ofembodiments 1-3, wherein the mixing well further comprises an exit portcomprising one or more openings formed in the second floor, wherein thefloor tapers downwardly toward the exit port.

Embodiment 5. The fluid sample processing cartridge of any one ofembodiments 1-4, wherein the driven mixing apparatus comprises a firstimpeller rotatably disposed within the mixing well and a gear configuredto be drivingly engaged by a mating gear of an instrument into which theliquid sample processing cartridge is inserted and to rotate the firstimpeller when engaged by the mating gear.

Embodiment 6. The fluid sample processing cartridge of any one ofembodiments 1-5, further comprising:

-   -   a lysis chamber containing a plurality of lysis beads, the lysis        chamber being formed in the substrate and disposed along the        channel connecting the mixing well and the sample well whereby        fluid flowing from the sample well to the mixing well will flow        through the lysis chamber; and    -   a bead mixer disposed at least partially within the lysis        chamber and constructed and arranged to agitate the lysis beads        and fluid flowing through the lysis chamber.

Embodiment 7. The fluid sample processing cartridge of embodiment 6,further comprising:

-   -   a first optical interface comprising an enlarged portion of the        channel connecting the lysis chamber to the sample well; and

a second optical interface comprising an enlarged portion of the channelconnecting the lysis chamber to the mixing well.

Embodiment 8. The fluid sample processing cartridge of embodiment 6 or7, wherein the bead mixer comprises:

a motor mounted within the substrate; and

a second impeller disposed within the lysis chamber and mounted on anoutput shaft of the motor.

Embodiment 9. The fluid sample processing cartridge of any one ofembodiments 6-8 wherein the lysis chamber includes a fluid inlet and afluid outlet and further comprises a mesh filter disposed over each ofthe fluid inlet and the fluid outlet and configured to retain the lysisbeads within the lysis chamber.

Embodiment 10. The fluid sample processing cartridge of any one ofembodiments 1-9, further comprising:

a pressure port formed in the substrate and configured to couple thesubstrate to an external fluid pressure source; and

a channel formed in the substrate connecting the pressure port to thesample well.

Embodiment 11. The fluid sample processing cartridge of embodiments1-10, further comprising:

-   -   a waste chamber formed in the substrate, the waste chamber being        in fluid communication with the mixing well via a channel formed        in the substrate;    -   a fluid exit port formed in the substrate, the fluid exit port        being in fluid communication with the mixing well via a channel        formed in the substrate;    -   a first externally actuatable control valve disposed within the        substrate and constructed and arranged to selectively permit or        prevent fluid flow from the mixing well to the waste chamber;        and    -   a second externally actuatable control valve disposed within the        substrate and constructed and arranged to selectively permit or        prevent fluid flow from the mixing well to the fluid exit port.

Embodiment 12. The fluid sample processing cartridge of embodiment 11,further comprising a capture chamber disposed along a channel connectingthe mixing well and the waste chamber

Embodiment 13. The fluid sample processing cartridge of embodiments1-12, further comprising:

-   -   a passive valve assembly disposed within the substrate and        constructed and arranged to be closed and prevent fluid flow        from the mixing well when pressure within the mixing well is not        higher than a threshold pressure and to open and permit fluid        flow from the mixing well when pressure within the mixing well        rises above the threshold pressure; and    -   a pressure port formed in the substrate and in pressure        communication with the passive valve assembly by a pressure        conduit formed in the substrate, wherein when the pressure port        is closed, pressure within the mixing well is allowed to reach        the threshold pressure that will open the passive valve assembly        and permit fluid flow from the mixing well, and when the        pressure port is open pressure within the mixing cannot not        reach the threshold pressure so the passive valve assembly is        closed.

Embodiment 14. The fluid sample processing cartridge of any one ofembodiments 1-13, further comprising a lance blister associated with thedeformable fluid chamber; the lance blister being connected orconnectable to the associated deformable fluid chamber and containing abead retained within the lance blister by a breakable septum, whereinthe lance blister is configured to collapse upon application of anexternal compression force to thereby push the bead through thebreakable septum.

Embodiment 15. The fluid sample processing cartridge of any one ofembodiments 1-14, further comprising an external shroud externallyenclosing at least a portion of the cartridge.

Embodiment 16. The fluid sample processing cartridge of any one ofembodiments 1-15, comprising a plurality of deformable fluid chambers,each of the fluid chambers containing one or more substances selectedfrom the group consisting of a lysis buffer, a wash buffer, an oil, arehydration buffer, target capture beads, and a binding buffer.

Embodiment 17. The fluid sample processing cartridge of any one ofembodiments 1-10, further comprising:

-   -   a first fluid exit port formed in the substrate, the first fluid        exit port being in fluid communication with the mixing well via        a channel formed in the substrate;    -   a second fluid exit port formed in the substrate; and    -   at least two deformable fluid chambers, one of the two        deformable fluid chambers being in fluid communication with the        mixing well via a channel formed in the substrate, and the other        of the two deformable fluid chambers being in fluid        communication with the second fluid exit port via a channel        formed in the substrate that is different from the channel        communicating the first fluid exit port with the mixing well.

Embodiment 18. The fluid sample processing cartridge of embodiment 17,wherein the deformable fluid chamber in fluid communication with themixing well contains a lysis buffer, a wash buffer, target capturebeads, or a binding buffer, and the deformable fluid chamber in fluidcommunication with the second fluid exit contains an oil or arehydration buffer.

Embodiment 19. A fluid sample processing cartridge comprising:

-   -   a) a sample preparation module comprising:    -   i) a substrate;    -   ii) a sample well formed in the substrate and configured to        receive a volume of fluid sample;    -   iii) a closure configured to be selectively placed over the        sample well;    -   iv) a first deformable fluid chamber supported on the substrate        and configured to hold a fluid therein when in an undeformed        state and to collapse upon application of an external        compression force to expel at least a portion of the fluid from        the first fluid chamber, the first deformable fluid chamber        being in fluid communication with the sample well via a channel        formed in the substrate;    -   v) a mixing well formed in the substrate, the mixing well being        in fluid communication with the sample well via a channel formed        in the substrate;    -   vi) a driven mixing apparatus disposed within the mixing well        and constructed and arranged to mix the contents of the mixing        well; and    -   vii) a first fluid exit port formed in the substrate, the first        fluid exit port being in fluid communication with the mixing        well via a channel formed in the substrate; and    -   b) a reaction module attached to the sample preparation module        and configured to receive a fluid from the sample preparation        module via the fluid exit port formed in the sample preparation        module, the reaction module comprising:

i) a top plate comprising

1) a top surface;

2) a raised wall at least partially circumscribing the top surface andin fluid sealing contact with a surface of the sample preparation moduleto form an interstitial space between the top surface and the surface ofthe sample preparation module;

3) a sample chamber fluidly coupled to the first fluid exit port of thesample preparation module;

4) a reagent chamber; and

5) a detection chamber; and

ii) a fluidic processing panel coupled to a bottom surface of the topplate and defining a reaction and processing space between the fluidicprocessing panel and the top plate, wherein the reaction and processingspace is open or openable to the sample chamber, the reaction chamber,and the detection chamber.

Embodiment 20. The fluid sample processing cartridge of embodiment 19,wherein the sample chamber of the reaction module includes an inlet portthrough which fluid sample enters the sample chamber and including a gapbetween the first fluid exit port of the sample preparation module andthe inlet port of the sample chamber, the gap being open to theinterstitial space.

Embodiment 21. The fluid sample processing cartridge of embodiment 19,wherein the first fluid exit port of the sample preparation modulecomprises an outlet channel formed through a frustoconical nipple.

Embodiment 22. The fluid sample processing cartridge of any one ofembodiments 19-21, wherein the reaction module further comprises anelectrosensor array disposed in each detection chamber.

Embodiment 23. The fluid sample processing cartridge of any one ofembodiments 19-22, wherein the top plate of the reaction module furthercomprises one or more bubble traps, each bubble trap comprising a bubblecapture hood open to the reaction and processing space and a ventopening open to the interstitial space.

Embodiment 24. The fluid sample processing cartridge of any one ofembodiments 19-23, wherein the sample preparation module furthercomprises:

a second deformable fluid chamber supported on the substrate andconfigured to hold a fluid therein when in an undeformed state and tocollapse upon application of an external compression force to expel atleast a portion the fluid from the fluid chamber; and

a second fluid exit port formed in the substrate, wherein the secondfluid exit port is in fluid communication with the second deformablefluid chamber via a channel formed in the substrate, and

wherein the reaction and processing space is fluidly coupled to thesecond fluid exit port of the sample preparation module.

Embodiment 25. The fluid sample processing cartridge of any one ofembodiments 19-24, wherein the mixing well comprises:

-   -   a peripheral wall and a floor defining a well; and    -   a fluid inlet snorkel extending up a side of the peripheral wall        extending from the channel communicating the mixing well to the        sample well and terminating below a top edge of the peripheral        wall.

Embodiment 26. The fluid sample processing cartridge of embodiment 25,wherein the fluid inlet snorkel extends up an outer surface of theperipheral wall and terminates at an opening formed in the peripheralwall.

Embodiment 27. The fluid sample processing cartridge of embodiment 25 or26, wherein the mixing well further comprises an exit port comprisingone or more openings formed in the floor of the mixing well, wherein thefloor tapers downwardly toward the exit port.

Embodiment 28. The fluid sample processing cartridge of any one ofembodiments 19-25, wherein the driven mixing apparatus comprises a firstimpeller rotatably disposed within the mixing well and a gear configuredto be drivingly engaged by a mating gear of an instrument into which theliquid sample processing cartridge is inserted and to rotate the firstimpeller when engaged by the mating gear.

Embodiment 29. The fluid sample processing cartridge of any one ofembodiments 19-28, wherein the sample preparation module furthercomprises:

-   -   a lysis chamber comprising a plurality of lysis beads, the lysis        chamber being formed in the substrate and disposed along the        channel connecting the mixing well and the sample well whereby        fluid flowing from the sample well to the mixing well will flow        through the lysis chamber; and    -   a bead mixer disposed at least partially within the lysis        chamber and constructed and arranged to agitate the lysis beads        and fluid flowing through the lysis chamber.

Embodiment 30. The fluid sample processing cartridge of embodiment 29,further comprising:

-   -   a first optical interface comprising an enlarged portion of the        channel connecting the lysis chamber to the sample well; and

a second optical interface comprising an enlarged portion of the channelconnecting the lysis chamber to the mixing well.

Embodiment 31. The fluid sample processing cartridge of embodiment 29 or30, wherein the bead mixer comprises:

a motor mounted within the substrate; and

a second impeller disposed within the lysis chamber and mounted on anoutput shaft of the motor.

Embodiment 32. The fluid sample processing cartridge of any one ofembodiments 29-31 wherein the lysis chamber includes a fluid inlet and afluid outlet and further comprises a mesh filter disposed over each ofthe fluid inlet and the fluid outlet and configured to retain the lysisbeads within the lysis chamber.

Embodiment 33. The fluid sample processing cartridge of any one ofembodiments 19-32, wherein the sample preparation module furthercomprises:

a pressure port formed in the substrate and configured to couple thesubstrate to an external fluid pressure source; and

a channel formed in the substrate connecting the pressure port to thesample well.

Embodiment 34. The fluid sample processing cartridge of embodiments19-33, wherein the sample preparation module further comprises:

-   -   a waste chamber formed in the substrate, the waste chamber being        in fluid communication with the mixing well via a channel formed        in the substrate;    -   a first externally actuatable control valve disposed within the        substrate and constructed and arranged to selectively permit or        prevent fluid flow from the mixing well to the waste chamber;        and    -   a second externally actuatable control valve disposed within the        substrate and constructed and arranged to selectively permit or        prevent fluid flow from the mixing well to the exit port.

Embodiment 35. The fluid sample processing cartridge of embodiment 34,wherein the sample preparation module further comprises a capturechamber disposed along a channel connecting the mixing well and thewaste chamber.

Embodiment 36. The fluid sample processing cartridge of embodiments19-35, wherein the sample preparation module further comprises:

-   -   a passive valve assembly disposed within the substrate and        constructed and arranged to be closed and prevent fluid flow        from the mixing well when pressure within the mixing well is not        higher than a threshold pressure and to open and permit fluid        flow from the mixing well when pressure within the mixing well        rises above the threshold pressure; and    -   a pressure port formed in the substrate and in pressure        communication with the passive valve assembly by a pressure        conduit formed in the substrate, wherein when the pressure port        is closed, pressure within the mixing well is allowed to reach        the threshold pressure that will open the passive valve assembly        and permit fluid flow from the mixing well, and when the        pressure port is open pressure within the mixing well cannot        reach the threshold pressure so the passive valve assembly is        closed.

Embodiment 37. The fluid sample processing cartridge of any one ofembodiments 19-36, wherein the sample preparation module furthercomprises a lance blister associated with the deformable fluid chamber;the lance blister being connected or connectable to the associateddeformable fluid chamber and containing a bead retained within the lanceblister by a breakable septum, wherein the lance blister is configuredto collapse upon application of an external compression force to therebypush the bead through the breakable septum.

Embodiment 38. The fluid sample processing cartridge of any one ofembodiments 19-37, further comprising an external shroud externallyenclosing at least a portion of the cartridge.

Embodiment 39. The fluid sample processing cartridge of any one ofembodiments 19-38, wherein the sample preparation module furthercomprises a plurality of deformable fluid chambers, each of the fluidchambers containing a substance selected from the group consisting of alysis buffer, a wash buffer, an oil, a rehydration buffer, targetcapture beads, and a binding buffer.

Embodiment 40. An instrument configured to process a fluid sampleprocessing cartridge including a deformable fluid chamber supported on aplanar substrate and configured to hold a fluid therein when in anundeformed state and to collapse upon application of an externalcompression force to expel at least a portion of the fluid from thefluid chamber, the instrument comprising:

-   -   a cartridge carriage assembly configured to receive and hold a        fluid sample processing cartridge inserted into the instrument;    -   a heating and control assembly adjacent the cartridge carriage        assembly and configured for movement with respect to the        cartridge carriage assembly between a first position not in        operative contact with the cartridge carried within the        cartridge carriage assembly and a second position in operative        contact with the cartridge carried within the cartridge carriage        assembly;    -   one or more movable magnet assemblies, each mounted for movement        with respect to the cartridge independently of the heating and        control assembly between a first position applying substantially        no magnetic force to the cartridge and a second position        applying magnetic force to corresponding discrete portions of        the cartridge;    -   a cam block assembly configured for powered movement and        operatively coupled to the heating and control assembly for        converting powered movement of the cam block assembly into        movement of the heating and control assembly with respect to the        cartridge carriage assembly between the first position of the        heating and control assembly and the second position of the        heating and control assembly and operatively coupled to the one        or more moveable magnet assemblies for converting powered        movement of the cam block assembly into movement of each magnet        assembly with respect to cartridge carriage assembly between the        first position of the magnet assembly and the second position of        the magnet assembly and;    -   e) a deformable chamber compression assembly configured to        selectively apply an external compression force to the        deformable fluid chamber to collapse the deformable chamber and        expel at least a portion of the fluid from the fluid chamber.

Embodiment 41. The instrument of embodiment 40, wherein the heating andcontrol assembly comprises:

one or more heater assemblies configured to apply a thermal gradient tocorresponding discrete portions of the cartridge when the heating andcontrol assembly is in the second position; and

a connector board including one or more electrical connector elementsconfigured to effect an electrical connection between the instrument andthe cartridge when the heating and control assembly is in the secondposition.

Embodiment 42. The instrument of embodiment 40 or 41, wherein thedeformable chamber compression assembly comprises:

a cam follower plate configured for powered movement in a firstdirection that is generally parallel to the plane of the substrate; and

a compression mechanism associated with the deformable chamber of thecartridge and configured to apply a force compressing the chamberagainst the substrate by movement in a second direction having acomponent that is generally normal to the plane of the substrate,

-   -   wherein the cam follower plate is operatively coupled to the        compression mechanism to convert movement of the cam follower        plate in the first direction into movement of the compression        mechanism in the second direction to thereby apply an external        compression force to the chamber.

Embodiment 43. The instrument of any one of embodiments 40-42, furthercomprising a pneumatic pump and a pneumatic port connected to thepneumatic pump, wherein the pneumatic port is configured to couple thepneumatic pump to a pressure port of the fluid sample processingcartridge when the cartridge is inserted into the instrument.

Embodiment 44. The instrument of any one of embodiments 40-43, furthercomprising an optical detector configured to detect fluid flow through apart of the fluid sample processing cartridge.

Embodiment 45. The instrument of any one of embodiments 40-42 whereinthe fluid sample processing cartridge includes a driven mixing apparatusincluding a drive gear, and wherein the instrument further comprises amixing motor assembly including a powered driving gear and moveablebetween a first position in which the driving gear is not engaged withthe drive gear of the driven mixing apparatus and a second position inwhich the driving gear is operatively engaged with the drive gear toactuate the driven mixing apparatus, and wherein the cam block assemblyis operatively coupled to the mixing motor assembly for convertingpowered movement of the cam block assembly into movement of the mixingmotor assembly between the first position of the mixing motor assemblyand the second position of the mixing motor assembly.

Embodiment 46. The instrument of any one of embodiments 41-45, furthercomprising a heater cooling assembly comprising:

-   -   a fan; and    -   a cooling duct configured to direct air flow from the fan to a        portion of one of the heater assemblies.

Embodiment 47. The instrument of any one of embodiments 40-46, whereinthe cartridge carriage assembly comprises:

a cartridge holder configured to hold a cartridge inserted therein;

a cartridge latch biased into a cartridge-latching position andconfigured to latch onto a cartridge inserted into the cartridge holderto retain the cartridge within the cartridge holder; and

a cartridge eject mechanism configured to automatically push a cartridgeat least partially out of the cartridge holder when the cartridge latchis released from a cartridge-latching position.

Embodiment 48. The instrument of any one of embodiments 41-47, whereinthe heating and control assembly comprises a support plate on which theone or more heater assemblies and the connector board are supported, thesupport plate being mounted in a constrain configuration preventinghorizontal movement of the support plate but permitting verticalmovement of the support plate to enable movement of the heating andcontrol assembly between its first and second positions.

Embodiment 49. The instrument of any one of embodiments 41-48, whereinone of the heater assemblies of the heating and control assemblycomprises a resistive heating element attached to the connector boardand a heat spreader comprising a thermally-conductive material thermallycoupled to the resistive heating element.

Embodiment 50. The instrument of any one of embodiments 41-49, whereinone of the heater assemblies of the heating and control assemblycomprises:

-   -   a thermoelectric element;    -   a heat spreader comprising a thermally-conductive material        thermally coupled to the thermoelectric element; and    -   a heat sink including a panel that is in thermal contact with        the thermoelectric element and a plurality of heat-dissipating        rods.

Embodiment 51. The instrument of any one of embodiments 41-50, whereinthe electrical connector elements of the connector board of the heatingand control assembly comprise a plurality of connector pin arrays, eachconnector pin array comprising a plurality of pogo pins.

Embodiment 52. The instrument of any one of embodiments 40-51, whereinone of the movable magnet assemblies comprises:

a magnet holder mounted on a spindle so as to be rotatable about thespindle between the first position and the second position of the magnetassembly;

a magnet supported on the magnet holder;

an actuator bracket extending from the magnet holder; and

a torsion spring configured to bias the magnet holder to a rotationalposition corresponding to the first position of the magnet assembly.

Embodiment 53. The instrument of any one of embodiments 40-52, whereinone of the movable magnet assemblies comprises:

a magnet holder frame mounted on a spindle so as to be rotatable aboutthe spindle between the first position and the second position of themagnet assembly;

a magnet array disposed within the magnet holder frame;

a focusing magnet disposed within an opening formed in the magnet holderframe and configured to focus magnetic forces of the magnet array;

an actuator bracket extending from the magnet holder frame; and

a torsion spring configured to bias the magnet holder frame to arotational position corresponding to the first position of the magnetassembly.

Embodiment 54. The instrument of embodiment 52 or 53, wherein the camblock assembly is operatively coupled to each movable magnet assembly bya magnet actuator coupled at one portion thereof to the cam blockassembly so as to be moveable by powered movement of the cam blockassembly and including a tab configured to be engageable with theactuator bracket of each magnet assembly as the magnet actuator is movedwith the cam block assembly to cause corresponding rotation of themagnet assembly from the first position to the second position.

Embodiment 55. The instrument of any one of embodiments 40-54, whereinthe cam block assembly comprises:

-   -   a cam frame;    -   a cam block motor coupled to the cam frame and configured to        effect powered movement of the cam frame; and    -   first and second cam rails attached to the cam frame, each of        the cam rails having two cam slots, wherein the cam block        assembly is operatively coupled to the heating and control        assembly by cam followers extending from the heating and control        assembly into the cam slots such that movement of the cam frame        and the cam rails with respect to the heating and control        assembly causes corresponding relative movement between the cam        followers and the cam slots to move the cam followers between        respective first segments of the cam slots corresponding to the        first position of the heating and control assembly and        respective second segments of the cam slots corresponding to the        second position of the heating and control assembly.

Embodiment 56. The instrument of embodiment 55, wherein the cam framecomprises:

a first longitudinal spar extending along one side of the heating andcontrol assembly;

a second longitudinal spar extending along an opposite side of theheating and control assembly; and

a cross spar extending between the first and second longitudinal spars,and wherein each cam rail is attached to one of the first and secondlongitudinal spars.

Embodiment 57. The instrument of any one of embodiments 42-56, whereinthe compression mechanism of the deformable chamber compression assemblycomprises:

-   -   a cam arm having a cam surface and mounted so as to be pivotable        about one end of the cam arm; and    -   a compression pad disposed at an opposite end of the cam arm,        wherein the cam arm is pivotable between a first position in        which the compression pad does not contact the associated        deformable chamber and a second position in which the        compression pad applies a compressive force to the associated        deformable chamber to at least partially collapse the chamber.

Embodiment 58. The instrument of embodiment 57, wherein the deformablechamber compression assembly further comprises a cam arm plate, and thecam arm of the compression mechanism is pivotably mounted within a slotformed in the cam arm plate for pivotable movement of the cam arm withrespect to the cam arm plate, and wherein the cam surface of the cam armprojects out of the slot above a surface of the cam arm plate, andwherein the cam follower plate is operatively coupled to the compressionmechanism by a cam follower element of the cam follower plate that isengaged with the cam surface of the compression mechanism duringmovement of the cam follower plate with respect to the cam arm plate tocause the cam arm to pivot from its first position to its secondposition.

Embodiment 59. The instrument of embodiment 58, wherein the cam followerplate comprises a cam groove that receives the cam surface of the camarm projecting above the surface of the cam arm plate, and the camfollower element comprises a follower ridge disposed within the camgroove that contacts the cam surface as the cam follower plate moveswith respect to the cam arm plate to cause the cam arm to pivot from itsfirst position to its second position.

Embodiment 60. The instrument of embodiments 59, comprising a pluralityof compression mechanisms each comprising a cam arm pivotably mountedwithin a slot formed in the cam arm plate and a cam arm surface, and thecam follower plate comprises a plurality of cam grooves, each cam groovebeing associated with at least one of the compression mechanisms andwherein each cam groove includes a follower ridge disposed within thecam groove that contacts the cam surface of the associated compressionmechanism as the cam follower plate moves with respect to the cam armplate to cause the cam arm of the associated compression mechanism topivot from its first position to its second position.

Embodiment 61. The instrument of any one of embodiments 42-60, whereinthe sample processing cartridge includes a plurality of deformable fluidchambers and wherein the deformable chamber compression assemblycomprises a plurality of compression mechanisms, each compressionmechanism being associated with one of the deformable fluid chambers,and wherein the cam follower plate is operatively coupled to thecompression mechanisms to convert movement of the cam follower plate inthe first direction into movement of each of the compression mechanismsin the second direction to thereby apply an external compression forceto each of the associated chambers in a specified sequence

Embodiment 62. The instrument of any one of embodiments 40-61, whereinthe fluid sample processing cartridge includes an externally-actuatablecontrol valve configured to selectively control fluid flow by permittingfluid flow through the valve when not externally actuated and preventingfluid flow through the valve when externally actuated, and wherein theinstrument further comprises a valve actuator compression mechanismassociated with the externally-actuatable control valve of the sampleprocessing cartridge and configured to actuate the associatedexternally-actuatable control valve by movement in a second directionhaving a component that is generally normal to the plane of thesubstrate, and wherein the cam follower plate is operatively coupled tothe valve actuator compression mechanism to convert movement of the camfollower plate in the first direction into movement of the valveactuator compression mechanism in the second direction to therebyactuate the associated externally-actuatable control valve.

While the subject matter of this disclosure has been described and shownin considerable detail with reference to certain illustrativeembodiments, including various combinations and sub-combinations offeatures, those skilled in the art will readily appreciate otherembodiments and variations and modifications thereof as encompassedwithin the scope of the present invention. Moreover, the descriptions ofsuch embodiments, combinations, and sub-combinations is not intended toconvey that the inventions requires features or combinations of featuresother than those expressly recited in the claims. Accordingly, the scopeof this disclosure is intended to include all modifications andvariations encompassed within the spirit and scope of the followingappended claims.

What is claimed is:
 1. A method of motivating a sample through acartridge, the method comprising: (a) motivating the sample on a firstsubstrate using microfluidics; and (b) motivating the sample on a secondsubstrate using electrowetting, thereby motivating a sample through acartridge, wherein (a) is not accomplished by electrowetting.
 2. Themethod of claim 1, further comprising, after (b), motivating the sampleinto an electrosensor array located on the second substrate.
 3. Themethod of claim 1, wherein (a) comprises pumping the sample from a firstlocation to a second location.
 4. The method of claim 1, wherein (a)comprises compressing a fluid-filled deformable compartment supported onthe first substrate, thereby generating pressure to move the sample. 5.The method of claim 1, wherein (a) comprises introducing magnetic beadsto the sample and applying a magnetic force to move the sample.
 6. Themethod of claim 1, wherein (a) comprises introducing negatively-chargedcapture beads to the first substrate to facilitate adsorption ofpositively-charged nucleic acids from the sample to a surface of thenegatively-charged capture beads, thereby moving the sample.
 7. Themethod of claim 1, further comprising introducing process fluids intothe first substrate by compressing a fluid-filled deformable compartmentsupported on the first substrate.
 8. The method of claim 1, furthercomprising: (c) retaining on the second substrate by electrowettingmanipulation an aliquot of the sample comprising: magnetic beads withDNA material bound thereto and wash solution; and (d) pulling themagnetic beads out of the wash solution using magnetic forces.
 9. Themethod of claim 1, wherein (a) comprises motivating the sample bypumping the sample, generating pressure in a microfluidic channel,applying magnetic forces to the sample, absorbing the sample, applyingrotary forces to the sample, or combinations thereof.
 10. The method ofclaim 1, wherein (a) comprises motivating the sample by pumping thesample, generating pressure in a microfluidic channel, applying magneticforces to the sample, absorbing the sample, and applying rotary forcesto the sample.
 11. The method of claim 1, further comprising, before(b), passing the sample through an interstitial space between the firstsubstrate and the second substrate, wherein the interstitial space formsa void.
 12. A method of motivating a sample through a cartridge, themethod comprising: (a) motivating the sample on a first substrate usinga first microfluidic technique; and (b) motivating the sample on asecond substrate using electrowetting, wherein the first microfluidictechnique and electrowetting are different, thereby motivating a samplethrough a cartridge.
 13. The method of claim 12, wherein (a) comprisesmotivating the sample by pumping the sample, generating pressure in amicrofluidic channel, applying magnetic forces to the sample, absorbingthe sample, applying rotary forces to the sample, or combinationsthereof.
 14. The method of claim 12, further comprising receiving asignal from an optical detector indicating an end of fluid flow throughan optical sensing chamber and in response to the signal deactivating arotary mixer motor.
 15. The method of claim 12, further comprising,after (b), on the second substrate, retaining magnetic target capturebeads and target analyte bound thereto by magnetic forces while flowinga remainder of contents of the sample into a waste chamber byelectrowetting manipulation.
 16. A method of motivating a sample througha cartridge, the method comprising: (a) motivating the sample on a firstsubstrate using microfluidics; (b) motivating the sample out of thefirst substrate into a sample outlet; (c) motivating the sample out ofthe sample outlet and into a sample inlet, thereby allowing gas bubblesto escape into an interstitial space; (d) motivating the sample out ofthe sample inlet and onto the second substrate; and (e) motivating thesample through the second substrate using electrowetting, therebymotivating a sample through a cartridge.
 17. The method of claim 16,wherein (a) comprises motivating the sample by pumping the sample,generating pressure in a microfluidic channel, applying magnetic forcesto the sample, absorbing the sample, applying rotary forces to thesample, or combinations thereof.
 18. The method of claim 16, furthercomprising, after (e), on the second substrate, motivating the sampleunder a bubble trap allowing bubbles to escape into the interstitialspace between the first substrate and the second substrate.
 19. Themethod of claim 16, further comprising applying a voltage to detectionelectrodes in an electrosensor array on the second substrate.
 20. Themethod of claim 16, further comprising introducing process fluids intothe second substrate by motivating fluid through an inlet channel on thefirst substrate, out an outlet port on the first substrate, and in aninlet port on the second substrate.
 21. A method for detecting apathogen in a sample, the method comprising: loading a sample into acartridge, the cartridge comprising a first substrate and a secondsubstrate; extracting nucleic acids from the sample in the firstsubstrate; transferring the sample to the second substrate; anddetecting a pathogen, if present, in the sample in the second substrate.